Methods and compositions for treating cancer using peptide nucleic acid-based agents

ABSTRACT

The present invention provides compositions and methods for treating cancer with peptide nucleic acid agents. In some embodiments, the present invention provides methods and compositions relating to peptide nucleic acid agents that target oncogenes. For example, the present invention provides compositions, including pharmaceutical compositions, comprising agents specific for BRAF V600E inhibition, or fragments or characteristic portions thereof. The present invention further provides various therapeutic and/or diagnostic methods of using BRAF V600E specific peptide nucleic acid agents and/or compositions.

CROSS REFERENCES TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 16/173,643, filed Oct. 29, 2018, which is acontinuation application of U.S. application Ser. No. 15/104,701, filedJun. 15, 2016, now U.S. Pat. No. 10,113,169, which is a National Stageof International Application No. PCT/US2014/070970, filed Dec. 17, 2014,which claims benefit to U.S. provisional application Ser. No. 61/920,289filed Dec. 23, 2013. The entire contents of the above applications areincorporated by reference as if recited in full herein.

BACKGROUND

The healthcare industry experiences a constant need to provide new andeffective therapies for treating patients battling cancer. The discoveryof novel compositions that deviate from traditional chemotherapeuticapproaches are aiding in the approach physicians use to prescribe courseof treatments for oncology patients. Particular effort is directedtoward compositions and methods for treating cancer using molecularbiological approaches rather than chemical approaches.

SUMMARY

The present invention provides new and effective compositions fortreating cancer. Among other things, the present invention recognizesthe source of a problem with conventional cancer treatments and providesthe insight that compounds which target gene (e.g., oncogene)expression, as described herein, are particularly useful in variouscontexts for treating cancer.

Among other things, the present disclosure demonstrates that peptidenucleic acid (PNA) agents that can specifically target genes, can enableimproved treatments for cancer. In some embodiments, use of peptidenucleic acid agents will enable improved methods for suppressing andtreating cancer within a clinical setting. Furthermore, peptide nucleicacids with terminal cationic and/or hydrophobic moieties that improvesolubility within cell membranes can be more effective at crossingcellular membranes than previous PNA-type agents and offer stabilizationtoward the anionic chromosomal target.

PNA agents are promising tools in the research and development of newdrugs to treat diseases such as cancer. The present disclosure providesimproved PNA agents, as well as technologies for designing, identifying,characterizing and/or using them, and compositions that include them.

Among other things, the present invention encompasses the recognitionthat one unmet need in use of available PNA-based drugs is thesuccessful delivery of agents across cellular membranes to the targetgene. For example, the ability to cross cell membranes is mediated bycationic/hydrophobic delivery peptides. Among other things, the presentinvention discloses PNA agents whose physio-chemical propertiescontribute to improved drug delivery across cell membranes (e.g.,relative to available PNA agents).

For example, the present disclosure demonstrates that cationicallycharged termini on PNA agents improve the ability to target non-promoterregions of genes, which are less open and exposed compared to promoterregions. Without wishing to be bound by any particular theory, thepresent disclosure proposes that stabilizing the cationically chargedlysine-derivatized PNA termini against the anionic DNA improves a PNA'sbinding kinetics to targets. These terminal modifications allowstabilization of the conjugate termini towards the chromosomal anionicphosphate esters due to cationic-anionic interaction as depicted in FIG.1A. This aids a PNA's strand-invading properties and allows it todisplace the complementary strand of its oncogene target. According tothe Zimm-Bragg statistical helical model, stabilization of theconfigurationally more labile portions (termini) towards helix formationis the most entropically disfavored step. Following stabilization of theterminus/termini, helix formation is comparatively immediate. This isalso consistent with the stochastic chain model.

PNA agents of the present disclosure are modified relative totraditional PNA agents through use of cationic/hydrophobic peptides; insome embodiments, such modified PNA agents show improved delivery acrosscell membranes relative to that observed with otherwise comparable PNAagents that do not include such cationic/hydrophobic peptides. Againwithout wishing to be bound by any particular theory, the presentdisclosure proposes that hydrophobic and cationic terminal peptidestogether facilitate passive transport of inventive PNA agents acrossmembranes. For example, in certain particular embodiments, hydrophobice-palmitoyl lysine termini are driven together by solvent exclusion, andthe PNA-peptide conjugate is intramolecularly further stabilized bypi-interacting nucleoside bases. In some embodiments, a PNA-peptidechain is compacted (i.e., displays a decreased radius of gyration)through such interactions, allowing it to more easily permeate amembrane (e.g., a lipid bilayer). Furthermore, it is hypothesized thatcationic-anionic interactions between PNA agents of the presentinvention and phospholipids cell membrane also facilitate the PNAagent's insertion into the membrane.

With respect to PNA agents, there is a thermodynamic equilibrium betweenstable binding within a cell membrane and dissociating from themembrane. Without wishing to be bound by any particular theory, it isenvisioned that the modifications of PNA agents of the present inventionease the kinetic barrier for membrane insertion of PNA agents so thatthe aforesaid equilibrium is more quickly achieved. This, in turn,accelerates transmembrane transport as depicted in FIG. 1B.

Without wishing to be bound by any particular theory, it is contemplatedthat a PNA-agent of the present invention exists in an equilibriumbetween folded and open states as depicted for example in FIG. 1C. Thefolded state lends itself well for cell membrane insertion, and theopen-coil state lends itself well for helical association withchromosomal targets for a better facilitated helix-coil transition.

The art has developed a variety of strategies for transportingoligonucleotides across cell membranes. The present invention providesimproved systems, permitting enhanced transport of provided PNAderivatives across cell membranes and intracellular delivery, andfurthermore facilitating binding of PNA agents to and targeting of lessexposed regions of DNA.

Embodiments of the present invention encompass the surprising discoverythat peptide nucleic acid agents with modified termini as describedherein can better cross cellular membranes and bind to target genes.According to some embodiments of the present invention, modified peptidenucleic acid agents specific for BRAF mutations, can suppresstranscription and ultimately translation of mutant BRAF protein as wellreduce viability of cells expressing the mutant protein.

Embodiments of the present invention encompass the surprising discoverythat peptide nucleic acid agents can bind to genes associated with adisease state.

In some embodiments, the invention provides PNA agents comprising: a PNAmoiety; a first cationic or hydrophobic moiety at a first end of the PNAmoiety; and a second cationic or hydrophobic moiety at a second end ofthe PNA moiety. In some embodiments, the first cationic moiety is orcomprises a peptide. In some embodiments, the first cationic peptidecomprises or consists of lysine residues. In some embodiments, at leastone lysine residue comprises a palmitoyl side chain moiety. In someembodiments, the first cationic peptide comprises amines.

In some embodiments, the invention provides PNA agents comprising: a PNAmoiety; a first cationic moiety and a first hydrophobic moiety at afirst end of the PNA moiety; and a second cationic moiety and a secondhydrophobic moiety at a second end of the PNA moiety. In someembodiments, the first cationic and/or hydrophobic moiety is orcomprises a peptide. In some embodiments, the second cationic and/orhydrophobic moiety is or comprises a peptide. In some embodiments, thefirst and/or second cationic peptide comprises one or more lysineresidues. In some embodiments, the first and/or second hydrophobicpeptide comprises one or more lysine residues. In some embodiments, atleast one lysine residue comprises a palmitoyl side chain moiety. Insome embodiments, at least one lysine residue at either end of the PNAmoiety comprises a palmitoyl side chain moiety. In some embodiments, thefirst and/or second cationic and/or hydrophobic peptide comprisesamines.

In some embodiments, the palmitoyl lysine is not attached to the PNAmoiety directly, but via one or more additional amino acids.

In some embodiments, the PNA agent has a sequence that does not formhairpin loops. In some embodiments, the PNA agent has a sequence thathas a tendency to not form hairpin loops. In some embodiments, the PNAagent has a sequence that contains less than 60% purines. In someembodiments, the first and/or second cationic and/or hydrophobic moietyis a targeting moiety in that the terminal cationic moieties moreeffectively align themselves with the DNA anionic phosphoribose. Thisallows them a greater statistical likelihood of finding the nucleic acidwith the sequence to which they are targeted. The terminalhydrophobic/cationic residues more effectively ease the PNA derivativethrough cell membranes. The termini likely associate intramolecularly by‘hydrophobic solvent exclusion,’ thus increasing the statistically highlikelihood of the termini being within proximity of each other. In someembodiments, the second cationic or hydrophobic moiety is or comprises acationic and/or hydrophobic peptide.

In some embodiments, the targeting moiety is at the PNA agent'sN-terminus. In some embodiments, the cationic peptide is at the PNAagent's C-terminus. In some embodiments, the first cationic orhydrophobic moiety is a targeting moiety in that the terminal cationicmoieties more effectively align themselves with the DNA anionicphosphoribose to allow a greater statistical likelihood of finding thenucleic acid with the sequence to which they are targeted and isattached at the PNA agent's N-terminus; and the second cationic orhydrophobic moiety is or comprises a cationic peptide that is attachedat the PNA agent's C-terminus.

In some embodiments, PNA agents comprise a sequence that targets a gene.In some embodiments, PNA agents comprise a sequence that targets a 13-20nucleotide sequence of a gene with 75% or greater complementarity. Insome embodiments, PNA agents comprise a nucleic acid whose length is atleast 14, 15, 16, 17, or 18 nucleotides and/or the complementarity is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In someembodiments, the PNA agent has a sequence that targets a non-promoterregion of a gene. In some embodiments, the gene is an oncogene. In someembodiments, the oncogene includes a mutant sequence element and the PNAagent has a sequence that targets a site comprising or consisting of themutant sequence element.

In some embodiments, the PNA component incorporates a sense (mRNA)sequence of a target gene.

In some embodiments, the PNA agent is targeted to a region of a BRAFoncogene comprising a mutation corresponding to a V600E mutation in aBRAF protein. In some embodiments, the PNA agent is targeted to a regionof a Gnaq gene comprising a mutation corresponding to a Q209L mutationin a Gnaq protein.

In some embodiments, the PNA agent is targeted to a region comprising atranslocation junction of an oncogene. In some embodiments, the PNAagent is targeted to a region of a MYB-NFIB translocation comprising ajunction of MYB and NFIB genes or fragments thereof. In someembodiments, the PNA agent is targeted to a region of a FUS-CHOPtranslocation comprising a junction of FUS and CHOP genes or fragmentsthereof.

In some embodiments, the PNA agent has a sequence that targets a site ina gene, which PNA agent is characterized in that, when a systemcomprising a cell that expresses the gene is exposed to the PNA agent,expression of the gene is reduced by an amount within the range of 20%to 90% suppression of normal activity when the PNA agent is present ascompared with otherwise comparable conditions when it is absent. In someembodiments, the PNA agent has a sequence that targets a site in a gene,which PNA agent is characterized in that, when a system comprising acell that expresses the gene is exposed to the PNA agent, expression ofthe gene is reduced by an amount within the range of 20% to 90% when thePNA agent is present as compared with otherwise comparable conditionswhen it is absent. In some embodiments, protein product is reduced toless than 50% expression. In some embodiments, the cell is a human cell.In some embodiments, the system is or comprises an animal. In someembodiments, the system is or comprises a primate. In some embodiments,the system is or comprises a human. In some embodiments, the system isor comprises a mouse. In some embodiments, the system is or comprises agenetically modified mouse. In some embodiments, the system is orcomprises a BRAF mouse. In some embodiments, the system is or comprisesthe cell in culture.

In some embodiments, the PNA moiety has a length within the range of13-18 nucleotides. In some embodiments, PNA moieties have palmitoyllysine attached to the termini. In some embodiments, PNA moieties havepalmitoyl lysine attached to both N- and C-termini. In some embodiments,PNA moieties have a delivery peptide length within the range of 8-12amino acids. In some embodiments, PNA-peptide conjugates areintramolecularly stabilized by pi-interacting nucleoside bases. In someembodiments, the radius of gyration of the PNA agent is decreased withinthe range of 25% to 50%. In some embodiments, PNA agents, when contactedwith a cell membrane, crosses the membrane 10 times as much as referencePNA agents lacking one or both terminal hydrophobic/cationic moieties.In some embodiments, gene suppression is approximately a magnitude moreeffective by employing cationic/hydrophobic Lys(palmitoyl)-Lys-Lysresidues on both termini in comparison to a standard deliverypeptide-PNA motif.

In some embodiments, a method for treating or reducing the risk of adisease, disorder, or condition comprising: administering to a subjectsusceptible to the disease, disorder, or condition a PNA agent isprovided. In some embodiments, the subject is suffering from orsusceptible to cancer. In some embodiments, the cancer is selected frommelanoma, ocular melanoma and/or sarcoma.

In some embodiments, methods of reducing expression of a target gene ina cell comprising: contacting a cell in which the target is expressedwith at least one PNA agent; determining a level or activity of thetarget in the cell when the PNA agent is present as compared with atarget reference level or activity observed under otherwise comparableconditions when it is absent; and classifying the at least one PNA agentas a target inhibitor if the level or activity of the target issignificantly reduced when the PNA agent is present as compared with thetarget reference level or activity are provided.

In some embodiments, a method for identifying and/or characterizing PNAagents for target inhibition comprising: contacting a system in which atarget is expressed with at least one PNA agent; determining a level oractivity of the target in the system when the PNA agent is present ascompared with a target reference level or activity observed underotherwise comparable conditions when it is absent; and classifying theat least one PNA agent as a target inhibitor if the level or activity ofthe target is significantly reduced when the PNA agent is present ascompared with the target reference level or activity is provided.

Any of the methods disclosed herein may include administering or usingany of the PNA agents disclosed herein.

In some embodiments, the level or activity of the target comprises atarget mRNA level. In some embodiments, the level or activity of thetarget comprises a target protein level. In some embodiments, the systemcomprises an in vitro system. In some embodiments, the system comprisesan in vivo system. In some embodiments, the system is or comprisescells.

In some embodiments, the level or activity of the target corresponds tocell viability. In some embodiments, a significant reduction in thelevel or activity of the target corresponds to a greater than 90%decrease in tumor cell viability.

In some embodiments, the cells comprise cancer cells. In someembodiments, the system comprises cells in cell culture. In someembodiments, the cells in cell culture comprise BRAF wild type cells. Insome embodiments, BRAF wild type cells comprise C918 cells. In someembodiments, the cells in cell culture comprise BRAF V600E melanomacells. In some embodiments, BRAF V600E melanoma cells are selected fromOCM1A uveal melanoma cells and/or SK-MEL 7 cutaneous melanoma cells.

In some embodiments, the system is or comprises tissue. In someembodiments, the system is or comprises an organism. In someembodiments, the level or activity of the target corresponds to survivalof the organism. In some embodiments, a significant reduction in thelevel or activity of the target comprises a greater than 50% increase insurvival of the organism.

In some embodiments, the organism comprises a mouse. In someembodiments, the mouse comprises a BRAF mouse. In some embodiments, themouse comprises a BRAF V600E mouse.

In some embodiments, a significant reduction in the level or activity ofthe target comprises a greater than 30% reduction of target activity. Insome embodiments, a significant reduction in the level or activity ofthe target comprises a greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% reduction of target levels. In some embodiments, a significantreduction in the level or activity of the target comprises a greaterthan two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold,eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, forty-fold,fifty-fold, sixty-fold, seventy-fold, eighty-fold, ninety-fold, onehundred-fold, two hundred-fold, three hundred-fold, four hundred-fold,five hundred-fold, six hundred-fold, seven hundred-fold, eighthundred-fold, nine hundred-fold, one thousand-fold, two thousand-fold,three thousand-fold, four thousand-fold, five thousand-fold, sixthousand-fold, seven thousand-fold, eight thousand-fold, ninethousand-fold, ten thousand-fold or more. In some embodiments, thereference level is a historical reference. In some embodiments, thehistorical reference is recorded in a tangible and/or computer-readablemedium.

In some embodiments, the target is a region comprising a point mutationin an oncogene. In some embodiments, the target is a region of a BRAFoncogene comprising a mutation corresponding to a V600E mutation in aBRAF protein. In some embodiments, the target is a region of a Gnaq genecomprising a mutation corresponding to a Q209L mutation in a Gnaqprotein.

In some embodiments, the target is a region comprising a translocationjunction of an oncogene. In some embodiments, the target is a MYB-NFIBtranslocation comprising a junction of MYB and NFIB genes or fragmentsthereof. In some embodiments, the target is a FUS-CHOP translocationcomprising a junction of FUS and CHOP genes or fragments thereof.

In some embodiments, a pharmaceutical composition comprising the PNAagent and pharmaceutically acceptable carrier is provided. In someembodiments, the pharmaceutical composition is formulated for directadministration into a target tissue. In some embodiments, thepharmaceutical composition is formulated for oral administration. Insome embodiments, the pharmaceutical composition is formulated forparenteral administration. In some embodiments, the pharmaceuticalcomposition is formulated for intradermal administration. In someembodiments, the pharmaceutical composition is formulated fortransdermal administration. In some embodiments, the pharmaceuticalcomposition is formulated for administration by inhalation.

In some embodiments, the pharmaceutical composition is or comprises aliquid. In some embodiments, the pharmaceutical composition is orcomprises a solid.

Additional features and advantages of the invention will be apparentfrom the following figures, definitions, detailed description and theclaims.

DESCRIPTION OF THE DRAWING

The Figures described below, that together make up the Drawing, are forillustration purposes only, not for limitation.

FIG. 1A shows schematic depictions of a PNA agent according to thepresent invention targeting DNA. FIG. 1B shows a schematic depiction ofa PNA agent according to the present invention crossing a cell membrane.FIG. 1C shows a schematic depiction of different states of a PNA agentaccording to the present invention.

FIGS. 2A-2B show data from a cell viability assay performed on melanomacell lines that had been treated with increasing doses of indicated PNAagents for 72 hours. Cell viability was measured and calculated relativeto the untreated cells. FIG. 2A depicts viability of BRAF V600E mutant(OCM1A, SK-MEL 7) and BRAF wild type (C918) cell lines treated with PNAI-292-3 L2LP NHAc. FIG. 2B depicts viability of the same melanoma celllines treated with PNA I-292-9 L2 NHAc. In both FIGS. 2A and 2B,suppression of cell viability and specificity for cell lines expressingthe target mutation was seen in OCM1A and SK-MEL 7 cells treated withthe PNA derivatives. Specificity was seen at lower doses in which cellviability of mutant cells is reduced to 20% or less with treatment ofPNA derivatives while wild type cell viability (C918) remains at around100%.

FIGS. 3A-3C depict BRAF mutant and BRAF wild type protein expression inmelanoma cells treated with 750 nM of PNA agent PNA I-292-3 L2LP NHAc(which incorporates an NLS delivery peptide) over the periods of timeindicated. FIG. 3A depicts the extent of mutant and total BRAF proteinexpression as measured by Western blots in C918 and OCM1A cells. Wildtype BRAF was found in both wild type and mutant cell lines, while theBRAF V600E mutant was only found in mutant cells. FIG. 3B depictsdecreased expression of BRAF V600E mutant protein in OCM1A (BRAF mutant)cell lines over 72 hours of treatment with PNA derivative. FIG. 3Cdepicts no significant reduction in total BRAF wild type protein over 72hours in either OCM1A (mutant) and C918 (wild type) cells treated withI-292-3 L2LP NHAc.

FIGS. 4A-4C depict BRAF mutant and BRAF wild type protein expression inmelanoma cells treated with 750 nM of PNA agent PNA I-292-9 L2 NHAc(which incorporates a TAT delivery peptide). FIG. 4A depicts the extentof mutant and total BRAF protein expression as measured by Western blotsin C918 and OCM1A cells. Wild type BRAF was found in both wild type andmutant cell lines, while the BRAF V600E mutant was only found in mutantcells. FIG. 4B depicts decreased expression of BRAF V600E mutant proteinin OCM1A (BRAF mutant) cell lines over 72 hours of treatment with PNAderivative. FIG. 4C depicts a slight reduction in total BRAF wild typeprotein over 72 hours in OCM1A (mutant) cells, but no significantreduction in C918 (wild type) cells treated with I-292-9 L2 NHAc PNA.

FIGS. 5A-5B depict mRNA expression in cells wild type for BRAF (C918)and mutant cell lines (OCM1A and SK-Mel 7). FIG. 5A depicts mutant BRAFV600E mRNA expression quantified by RT-PCR. BRAF V600E mRNA expressionwas significantly decreased in OCM1A cells when treated with PNA agentI-292-3 L2LP NHAc. FIG. 5B depicts both BRAF V600E and total BRAF mRNAexpression in SK-Mel 7 cells quantified with RT-PCR. In SK-Mel 7 cells,BRAF V600E mRNA expression was decreased significantly after treatmentwith PNA agent I-292-3 L2LP NHAc. There was no decrease in total BRAFmRNA expression.

FIGS. 6A-6B show data from xenograft mouse model studies employing OCM1Acells. FIG. 6A shows 50% tumor volume regression after three 50 mg/kg IPdoses of PNA agent I-292-3 L2LP NHAc given on days 5, 17, 20, and 22.Tumor shrinkage begins to increase more than a week following the lastdose. FIG. 6B shows, that treated mice exhibit no side effects as therewas no weight loss or changes in daily activity over a the entire lengthof the treatment shown in FIG. 6A.

DEFINITIONS

Agent: The term “agent” as used herein may refer to a compound or entityof any chemical class including, for example, polypeptides, nucleicacids, saccharides, lipids, small molecules, metals, or combinationsthereof. As will be clear from context, in some embodiments, an agentcan be or comprise a cell or organism, or a fraction, extract, orcomponent thereof. In some embodiments, an agent is agent is orcomprises a natural product in that it is found in and/or is obtainedfrom nature. In some embodiments, an agent is or comprises one or moreentities that is man-made in that it is designed, engineered, and/orproduced through action of the hand of man and/or is not found innature. In some embodiments, an agent may be utilized in isolated orpure form; in some embodiments, an agent may be utilized in crude form.In some embodiments, potential agents are provided as collections orlibraries, for example that may be screened to identify or characterizeactive agents within them. Some particular embodiments of agents thatmay be utilized in accordance with the present invention include smallmolecules, antibodies, antibody fragments, aptamers, siRNAs, shRNAs,DNA/RNA hybrids, antisense oligonucleotides, ribozymes, peptides,peptide mimetics, peptide nucleic acids, small molecules, etc. In someembodiments, an agent is or comprises a polymer. In some embodiments, anagent is not a polymer and/or is substantially free of any polymer. Insome embodiments, an agent contains at least one polymeric moiety. Insome embodiments, an agent lacks or is substantially free of anypolymeric moiety.

Affinity: As is known in the art, “affinity” is a measure of thetightness with a particular ligand (e.g., an HA polypeptide) binds toits partner (e.g., an HA receptor). Affinities can be measured indifferent ways. In some embodiments, affinity is measured by aquantitative assay (e.g., glycan binding assays). In some suchembodiments, binding partner concentration (e.g., HA receptor, glycan,etc.) may be fixed to be in excess of ligand (e.g., an HA polypeptide)concentration so as to mimic physiological conditions (e.g., viral HAbinding to cell surface glycans). Alternatively or additionally, in someembodiments, binding partner (e.g., HA receptor, glycan, etc.)concentration and/or ligand (e.g., an HA polypeptide) concentration maybe varied. In some such embodiments, affinity (e.g., binding affinity)may be compared to a reference (e.g., a wild-type HA that mediatesinfection of a humans) under comparable conditions (e.g.,concentrations).

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H2N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an 1-amino acid. “Standardamino acid” refers to any of the twenty standard 1-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide. Animal: As used herein, the term “animal” refersto any member of the animal kingdom. In some embodiments, “animal”refers to humans, of either sex and at any stage of development. In someembodiments, “animal” refers to non-human animals, at any stage ofdevelopment. In some embodiments, the non-human animal is a mammal(e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, asheep, cattle, a primate, and/or a pig). In some embodiments, animalsinclude, but are not limited to, mammals, birds, reptiles, amphibians,fish, insects, and/or worms. In some embodiments, the animal issusceptible to infection by HCV. In some embodiments, an animal may be atransgenic animal, genetically engineered animal, and/or a clone.

Antibody agent: As used herein, the term “antibody agent” refers to anagent that specifically binds to a particular antigen. In someembodiments, the term encompasses any polypeptide with immunoglobulinstructural elements sufficient to confer specific binding. Suitableantibody agents include, but are not limited to, human antibodies,primatized antibodies, chimeric antibodies, bi-specific antibodies,humanized antibodies, conjugated antibodies (i.e., antibodies conjugatedor fused to other proteins, radiolabels, cytotoxins), Small ModularImmunoPharmaceuticals (“SMIPs™”), single chain antibodies, cameloidantibodies, and antibody fragments. As used herein, the term “antibodyagent” also includes intact monoclonal antibodies, polyclonalantibodies, single domain antibodies (e.g., shark single domainantibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies(e.g. bi-specific antibodies) formed from at least two intactantibodies, and antibody fragments so long as they exhibit the desiredbiological activity. In some embodiments, the term encompasses stapledpeptides. In some embodiments, the term encompasses one or moreantibody-like binding peptidomimetics. In some embodiments, the termencompasses one or more antibody-like binding scaffold proteins. In someembodiments, the term encompasses monobodies or adnectins. In someembodiments, an antibody agent is or comprises a polypeptide whose aminoacid sequence includes one or more structural elements recognized bythose skilled in the art as a complementarity determining region (CDR);in some embodiments an antibody agent is or comprises a polypeptidewhose amino acid sequence includes at least one CDR (e.g., at least oneheavy chain CDR and/or at least one light chain CDR) that issubstantially identical to one found in a reference antibody. In someembodiments an included CDR is substantially identical to a referenceCDR in that it is either identical in sequence or contains between 1-5amino acid substitutions as compared with the reference CDR. In someembodiments an included CDR is substantially identical to a referenceCDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with thereference CDR. In some embodiments an included CDR is substantiallyidentical to a reference CDR in that it shows at least 96%, 96%, 97%,98%, 99%, or 100% sequence identity with the reference CDR. In someembodiments an included CDR is substantially identical to a referenceCDR in that at least one amino acid within the included CDR is deleted,added, or substituted as compared with the reference CDR but theincluded CDR has an amino acid sequence that is otherwise identical withthat of the reference CDR. In some embodiments an included CDR issubstantially identical to a reference CDR in that 1-5 amino acidswithin the included CDR are deleted, added, or substituted as comparedwith the reference CDR but the included CDR has an amino acid sequencethat is otherwise identical to the reference CDR. In some embodiments anincluded CDR is substantially identical to a reference CDR in that atleast one amino acid within the included CDR is substituted as comparedwith the reference CDR but the included CDR has an amino acid sequencethat is otherwise identical with that of the reference CDR. In someembodiments an included CDR is substantially identical to a referenceCDR in that 1-5 amino acids within the included CDR are deleted, added,or substituted as compared with the reference CDR but the included CDRhas an amino acid sequence that is otherwise identical to the referenceCDR. In some embodiments, an antibody agent is or comprises apolypeptide whose amino acid sequence includes structural elementsrecognized by those skilled in the art as an immunoglobulin variabledomain. In some embodiments, an antibody agent is a polypeptide proteinhaving a binding domain which is homologous or largely homologous to animmunoglobulin-binding domain.

Antagonist: As used herein, the term “antagonist” refers to an agentthat i) inhibits, decreases or reduces the effects of another agent, forexample that inactivates a nucleic acid; and/or ii) inhibits, decreases,reduces, or delays one or more biological events, for example,expression of one or more nucleic acids or stimulation of one or morebiological pathways. Antagonists may be or include agents of anychemical class including, for example, small molecules, polypeptides,nucleic acids, carbohydrates, lipids, metals, and/or any other entitythat shows the relevant inhibitory activity. An antagonist may be direct(in which case it exerts its influence directly upon the receptor) orindirect (in which case it exerts its influence by other than binding tothe receptor; e.g., altering expression or translation of the receptor;altering signal transduction pathways that are directly activated by thereceptor, altering expression, translation or activity of an agonist ofthe receptor).

Antibody polypeptide: As used herein, the terms “antibody polypeptide”or “antibody”, or “antigen-binding fragment thereof”, which may be usedinterchangeably, refer to polypeptide(s) capable of binding to anepitope. In some embodiments, an antibody polypeptide is a full-lengthantibody, and in some embodiments, is less than full length but includesat least one binding site (comprising at least one, and preferably atleast two sequences with structure of antibody “variable regions”). Insome embodiments, the term “antibody polypeptide” encompasses anyprotein having a binding domain which is homologous or largelyhomologous to an immunoglobulin-binding domain. In some embodiments,“antibody polypeptides” encompasses polypeptides having a binding domainthat shows at least 99% identity with an immunoglobulin binding domain.In some embodiments, “antibody polypeptide” is any protein having abinding domain that shows at least 70%, 80%, 85%, 90%, or 95% identitywith an immunoglobulin binding domain, for example a referenceimmunoglobulin binding domain. An included “antibody polypeptide” mayhave an amino acid sequence identical to that of an antibody that isfound in a natural source. Antibody polypeptides in accordance with thepresent invention may be prepared by any available means including, forexample, isolation from a natural source or antibody library,recombinant production in or with a host system, chemical synthesis,etc., or combinations thereof. An antibody polypeptide may be monoclonalor polyclonal. An antibody polypeptide may be a member of anyimmunoglobulin class, including any of the human classes: IgG, IgM, IgA,IgD, and IgE. In some embodiments, an antibody may be a member of theIgG immunoglobulin class. As used herein, the terms “antibodypolypeptide” or “characteristic portion of an antibody” are usedinterchangeably and refer to any derivative of an antibody thatpossesses the ability to bind to an epitope of interest. In someembodiments, the “antibody polypeptide” is an antibody fragment thatretains at least a significant portion of the full-length antibody'sspecific binding ability. Examples of antibody fragments include, butare not limited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fdfragments. Alternatively or additionally, an antibody fragment maycomprise multiple chains that are linked together, for example, bydisulfide linkages. In some embodiments, an antibody polypeptide may bea human antibody. In some embodiments, the antibody polypeptides may bea humanized. Humanized antibody polypeptides include may be chimericimmunoglobulins, immunoglobulin chains or antibody polypeptides (such asFv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. In general, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary-determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity.

Antigen: An “antigen” is a molecule or entity to which an antibodybinds. In some embodiments, an antigen is or comprises a polypeptide orportion thereof. In some embodiments, an antigen is a portion of aninfectious agent that is recognized by antibodies. In some embodiments,an antigen is an agent that elicits an immune response; and/or (ii) anagent that is bound by a T cell receptor (e.g., when presented by an MHCmolecule) or to an antibody (e.g., produced by a B cell) when exposed oradministered to an organism. In some embodiments, an antigen elicits ahumoral response (e.g., including production of antigen-specificantibodies) in an organism; alternatively or additionally, in someembodiments, an antigen elicits a cellular response (e.g., involvingT-cells whose receptors specifically interact with the antigen) in anorganism. It will be appreciated by those skilled in the art that aparticular antigen may elicit an immune response in one or severalmembers of a target organism (e.g., mice, rabbits, primates, humans),but not in all members of the target organism species. In someembodiments, an antigen elicits an immune response in at least about25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the members of a targetorganism species. In some embodiments, an antigen binds to an antibodyand/or T cell receptor, and may or may not induce a particularphysiological response in an organism. In some embodiments, for example,an antigen may bind to an antibody and/or to a T cell receptor in vitro,whether or not such an interaction occurs in vivo. In general, anantigen may be or include any chemical entity such as, for example, asmall molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid,a polymer other than a biologic polymer (e.g., other than a nucleic acidor amino acid polymer) etc. In some embodiments, an antigen is orcomprises a polypeptide. In some embodiments, an antigen is or comprisesa glycan. Those of ordinary skill in the art will appreciate that, ingeneral, an antigen may be provided in isolated or pure form, oralternatively may be provided in crude form (e.g., together with othermaterials, for example in an extract such as a cellular extract or otherrelatively crude preparation of an antigen-containing source). In someembodiments, antigens utilized in accordance with the present inventionare provided in a crude form. In some embodiments, an antigen is orcomprises a recombinant antigen.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In some embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system (e.g., cell culture, organism, etc.). For instance, asubstance that, when administered to an organism, has a biologicaleffect on that organism, is considered to be biologically active. Insome embodiments, where a protein or polypeptide is biologically active,a portion of that protein or polypeptide that shares at least onebiological activity of the protein or polypeptide is typically referredto as a “biologically active” portion.

Characteristic portion: As used herein, the term a “characteristicportion” of a substance, in the broadest sense, is one that shares somedegree of sequence or structural identity with respect to the wholesubstance. In some embodiments, a characteristic portion shares at leastone functional characteristic with the intact substance. For example, a“characteristic portion” of a protein or polypeptide is one thatcontains a continuous stretch of amino acids, or a collection ofcontinuous stretches of amino acids, that together are characteristic ofa protein or polypeptide. In some embodiments, each such continuousstretch generally contains at least 2, 5, 10, 15, 20, 50, or more aminoacids. In general, a characteristic portion of a substance (e.g., of aprotein, antibody, etc.) is one that, in addition to the sequence and/orstructural identity specified above, shares at least one functionalcharacteristic with the relevant intact substance; epitope-bindingspecificity is one example. In some embodiments, a characteristicportion may be biologically active.

Combination therapy: The term “combination therapy”, as used herein,refers to those situations in which two or more different pharmaceuticalagents for the treatment of disease are administered in overlappingregimens so that the subject is simultaneously exposed to at least twoagents. In some embodiments, the different agents are administeredsimultaneously. In some embodiments, the administration of one agentoverlaps the administration of at least one other agent. In someembodiments, the different agents are administered sequentially suchthat the agents have simultaneous biologically activity with in asubject.

Detection entity: The term “detection entity” as used herein refers toany element, molecule, functional group, compound, fragments thereof ormoiety that facilitates detection of an agent (e.g., an antibody) towhich it is joined. Examples of detection entities include, but are notlimited to: various ligands, radionuclides (e.g., ³H, ¹⁴C, ¹⁸F, ¹⁹F,³²P, ³⁵S, ¹³⁵I, ¹²⁵I, ¹²³I, ⁶⁴Cu, ¹⁸⁷Re, ¹¹¹In, ⁹⁰Y, ^(99m)Tc, ¹⁷⁷Lu,⁸⁹Zr etc.), fluorescent dyes (for specific exemplary fluorescent dyes,see below), chemiluminescent agents (such as, for example, acridinumesters, stabilized dioxetanes, and the like), bioluminescent agents,spectrally resolvable inorganic fluorescent semiconductors nanocrystals(i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper,platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes (forspecific examples of enzymes, see below), colorimetric labels (such as,for example, dyes, colloidal gold, and the like), biotin, dioxigenin,haptens, and proteins for which antisera or monoclonal antibodies areavailable.

Diagnostic information: As used herein, diagnostic information orinformation for use in diagnosis is any information that is useful indetermining whether a patient has a disease or condition and/or inclassifying the disease or condition into a phenotypic category or anycategory having significance with regard to prognosis of the disease orcondition, or likely response to treatment (either treatment in generalor any particular treatment) of the disease or condition. Similarly,diagnosis refers to providing any type of diagnostic information,including, but not limited to, whether a subject is likely to have adisease or condition (such as cancer), state, staging or characteristicof the disease or condition as manifested in the subject, informationrelated to the nature or classification of a tumor, information relatedto prognosis and/or information useful in selecting an appropriatetreatment. Selection of treatment may include the choice of a particulartherapeutic (e.g., chemotherapeutic) agent or other treatment modalitysuch as surgery, radiation, etc., a choice about whether to withhold ordeliver therapy, a choice relating to dosing regimen (e.g., frequency orlevel of one or more doses of a particular therapeutic agent orcombination of therapeutic agents), etc.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic compositionto be administered to a subject. Each unit contains a predeterminedquantity of active material (e.g., a therapeutic agent). In someembodiments, the predetermined quantity is one that has been correlatedwith a desired therapeutic effect when administered as a dose in adosing regimen. Those of ordinary skill in the art appreciate that thetotal amount of a therapeutic composition or agent administered to aparticular subject is determined by one or more attending physicians andmay involve administration of multiple dosage forms.

Dosing regimen: A “dosing regimen” (or “therapeutic regimen”), as thatterm is used herein, is a set of unit doses (typically more than one)that are administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regimen, which may involve one or more doses. In someembodiments, a dosing regimen comprises a plurality of doses each ofwhich are separated from one another by a time period of the samelength; in some embodiments, a dosing regimen comprises a plurality ofdoses and at least two different time periods separating individualdoses. In some embodiments, a dosing regimen is or has been correlatedwith a desired therapeutic outcome, when administered across apopulation of patients.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end formation); (3) translation of an RNA into a polypeptide orprotein; and/or (4) posttranslational modification of a polypeptide orprotein.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized. A biological molecule may havetwo functions (i.e., bifunctional) or many functions (i.e.,multifunctional).

Gene: As used herein, the term “gene” has its meaning as understood inthe art. In some embodiments, the term “gene” may include generegulatory sequences (e.g., promoters, enhancers, etc.) and/or intronsequences. In some embodiments, the term refers to nucleic acids that donot encode proteins but rather encode functional RNA molecules such astRNAs, RNAi-inducing agents, etc. Alternatively or additionally, in someembodiments, the term “gene”, as used in the present application, refersto a portion of a nucleic acid that encodes a protein. Whether the termencompasses other sequences (e.g., non-coding sequences, regulatorysequences, etc) will be clear from context to those of ordinary skill inthe art.

Gene product or expression product: As used herein, the term “geneproduct” or “expression product” generally refers to an RNA transcribedfrom the gene (pre- and/or post-processing) or a polypeptide (pre-and/or post-modification) encoded by an RNA transcribed from the gene.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g., between polypeptidemolecules. In some embodiments, polymeric molecules such as antibodiesare considered to be “homologous” to one another if their sequences areat least 80%, 85%, 90%, 95%, or 99% identical. In some embodiments,polymeric molecules are considered to be “homologous” to one another iftheir sequences are at least 80%, 85%, 90%, 95%, or 99% similar.

Lysine or lysine residue: As used herein, the term “lysine” or “lysineresidue” refers to the basic amino acid residue and its derivatives.Such derivatives include those lysine residues with side chainmodifications. Lysine derivatives include e-palmitoyl lysine orLys(palmitoyl-(dLys)2. Marker: A marker, as used herein, refers to anagent whose presence or level is a characteristic of a particular tumoror metastatic disease thereof. For example, in some embodiments, theterm refers to a gene expression product that is characteristic of aparticular tumor, tumor subclass, stage of tumor, etc. Alternatively oradditionally, in some embodiments, a presence or level of a particularmarker correlates with activity (or activity level) of a particularsignaling pathway, for example that may be characteristic of aparticular class of tumors. The statistical significance of the presenceor absence of a marker may vary depending upon the particular marker. Insome embodiments, detection of a marker is highly specific in that itreflects a high probability that the tumor is of a particular subclass.Such specificity may come at the cost of sensitivity (i.e., a negativeresult may occur even if the tumor is a tumor that would be expected toexpress the marker). Conversely, markers with a high degree ofsensitivity may be less specific that those with lower sensitivity.According to the present invention a useful marker need not distinguishtumors of a particular subclass with 100% accuracy.

Mutant: As used herein, the term “mutant” refers to any alteration in anucleic acid (or optionally genetic) sequence compared to itsnaturally-occurring counterpart. Mutant may also refer to the geneproduct (such as a protein), cells, or organism that possesses themutated gene. Nucleic acid sequences possessing mutations can also bereferred to as mutant sequence elements.

Non-promoter region: As used herein, the term “non-promoter region”refers to those section(s) of genes that are not sites of initiation oftranscription. Unlike promoter regions, non-promoter regions tend to beclosed off and less accessible to other elements.

Oncogene: As used herein, the term “oncogene” refers to those geneswhose products are associated with causing cancer, dysplasia,hyperplasia, etc. in an organism. Oncogenes of the present disclosuremay include, but are not limited to: ABL1, ABL2, ALK, AKT1, AKT2, ATF1,BCL11A, BCL2, BLC3, BCL6, BCR, BRAF, CARD11, CBLB, CBLC, CCND1, CCND2,CCND3, CDX2, CTNNB1, DDB2, DDIT3, DDX6, DEK, EGFR, ELK4, ERBB2, ETV4,ETV6, EVIL EWSR1, FEV, FGFR1, FGFR1OP, FGFR2, FUS, GOLGA5, HMGA1, HMGA2,HRAS, IRF4, IDHL IDH2, JUN, KIT, KRAS, LCK, LMO2, MAF, MAFB, MAML2,MDM2, MET, MITF, MLL, MPL, MYB, MYC, MYCL1, MYCN, NCOA4, NFKB2, NRAS,NTRK1, NUP214, PAX8, PDGFB, PIK3CA, PIM1, PLAG1, PPARG, PTPN11, RAFT,REL, RET, ROS1, SMO, SS18, TCL1A, TET2, TFG, TLX1, TPR, and USP6.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition is or may be administered,e.g., for experimental, diagnostic, prophylactic, cosmetic, and/ortherapeutic purposes. Typical patients include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and/or humans). In someembodiments, a patient is a human. In some embodiments, a patient issuffering from or susceptible to one or more disorders or conditions. Insome embodiments, a patient displays one or more symptoms of a disorderor condition. In some embodiments, a patient has been diagnosed with oneor more disorders or conditions. In some embodiments, the disorder orcondition is or includes cancer, or presence of one or more tumors. Insome embodiments, such cancer or tumor is or comprises a cancer of theprostate, or tumor in the prostate. In some embodiments, the disorder orcondition is metastatic cancer. In some embodiments, the disorder orcondition is melanoma.

Peptide: The term “peptide” refers to two or more amino acids joined toeach other by peptide bonds or modified peptide bonds. In someembodiments, “peptide” refers to a polypeptide having a length of lessthan about 100 amino acids, less than about 50 amino acids, less than 20amino acids, or less than 10 amino acids.

Peptide nucleic acid: The term “peptide nucleic acid” refers tosynthetic polymers similar to DNA or RNA, but lacking deoxyribose andribose sugar backbones, respectively. Peptide nucleic acids possess abackbone composed of repeating N-(2-aminoethyl)-glycine units linked bypeptide bonds. Purine and pyrimidine bases are linked to the backbone bya methylene bridge and carbonyl group.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to an active agent, formulated together with one ormore pharmaceutically acceptable carriers. In some embodiments, activeagent is present in unit dose amount appropriate for administration in atherapeutic regimen that shows a statistically significant probabilityof achieving a predetermined therapeutic effect when administered to arelevant population. In some embodiments, pharmaceutical compositionsmay be specially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

Polypeptide: As used herein, a “polypeptide”, generally speaking, is astring of at least two amino acids attached to one another by a peptidebond. In some embodiments, a polypeptide may include at least 3-5 aminoacids, each of which is attached to others by way of at least onepeptide bond. Those of ordinary skill in the art will appreciate thatpolypeptides sometimes include “non-natural” amino acids or otherentities that nonetheless are capable of integrating into a polypeptidechain, optionally.

Prognostic and predictive information: As used herein, the termsprognostic and predictive information are used interchangeably to referto any information that may be used to indicate any aspect of the courseof a disease or condition either in the absence or presence oftreatment. Such information may include, but is not limited to, theaverage life expectancy of a patient, the likelihood that a patient willsurvive for a given amount of time (e.g., 6 months, 1 year, 5 years,etc.), the likelihood that a patient will be cured of a disease, thelikelihood that a patient's disease will respond to a particular therapy(wherein response may be defined in any of a variety of ways).Prognostic and predictive information are included within the broadcategory of diagnostic information.

Promoter: As used herein, the term “promoter” refers to regions of DNAthat serve as initiation sites for transcription of a particular gene.Promoter sequences are often open/unraveled and await binding to otherelements.

Protein: As used herein, the term “protein” refers to a polypeptide(i.e., a string of at least 3-5 amino acids linked to one another bypeptide bonds). Proteins may include moieties other than amino acids(e.g., may be glycoproteins, proteoglycans, etc.) and/or may beotherwise processed or modified. In some embodiments “protein” can be acomplete polypeptide as produced by and/or active in a cell (with orwithout a signal sequence); in some embodiments, a “protein” is orcomprises a characteristic portion such as a polypeptide as produced byand/or active in a cell. In some embodiments, a protein includes morethan one polypeptide chain. For example, polypeptide chains may belinked by one or more disulfide bonds or associated by other means. Insome embodiments, proteins or polypeptides as described herein maycontain L-amino acids, D-amino acids, or both, and/or may contain any ofa variety of amino acid modifications or analogs known in the art.Useful modifications include, e.g., terminal acetylation, amidation,methylation, etc. In some embodiments, proteins or polypeptides maycomprise natural amino acids, non-natural amino acids, synthetic aminoacids, and/or combinations thereof. In some embodiments, proteins are orcomprise antibodies, antibody polypeptides, antibody fragments,biologically active portions thereof, and/or characteristic portionsthereof.

Response: As used herein, a response to treatment may refer to anybeneficial alteration in a subject's condition that occurs as a resultof or correlates with treatment. Such alteration may includestabilization of the condition (e.g., prevention of deterioration thatwould have taken place in the absence of the treatment), amelioration ofsymptoms of the condition, and/or improvement in the prospects for cureof the condition, etc. It may refer to a subject's response or to atumor's response. Tumor or subject response may be measured according toa wide variety of criteria, including clinical criteria and objectivecriteria. Techniques for assessing response include, but are not limitedto, clinical examination, positron emission tomatography, chest X-ray CTscan, MRI, ultrasound, endoscopy, laparoscopy, presence or level oftumor markers in a sample obtained from a subject, cytology, and/orhistology. Many of these techniques attempt to determine the size of atumor or otherwise determine the total tumor burden. Methods andguidelines for assessing response to treatment are discussed in Therasseet. al., “New guidelines to evaluate the response to treatment in solidtumors”, European Organization for Research and Treatment of Cancer,National Cancer Institute of the United States, National CancerInstitute of Canada, J. Natl. Cancer Inst., 2000, 92(3):205-216. Theexact response criteria can be selected in any appropriate manner,provided that when comparing groups of tumors and/or patients, thegroups to be compared are assessed based on the same or comparablecriteria for determining response rate. One of ordinary skill in the artwill be able to select appropriate criteria.

Sample: As used herein, a sample obtained from a subject may include,but is not limited to, any or all of the following: a cell or cells, aportion of tissue, blood, serum, ascites, urine, saliva, and other bodyfluids, secretions, or excretions. The term “sample” also includes anymaterial derived by processing such a sample. Derived samples mayinclude nucleotide molecules or polypeptides extracted from the sampleor obtained by subjecting the sample to techniques such as amplificationor reverse transcription of mRNA, etc.

Specific binding: As used herein, the terms “specific binding” or“specific for” or “specific to” refer to an interaction (typicallynon-covalent) between a target entity (e.g., a target protein orpolypeptide) and a binding agent (e.g., an antibody, such as a providedantibody). As will be understood by those of ordinary skill, aninteraction is considered to be “specific” if it is favored in thepresence of alternative interactions. In some embodiments, aninteraction is typically dependent upon the presence of a particularstructural feature of the target molecule such as an antigenicdeterminant or epitope recognized by the binding molecule. For example,if an antibody is specific for epitope A, the presence of a polypeptidecontaining epitope A or the presence of free unlabeled A in a reactioncontaining both free labeled A and the antibody thereto, will reduce theamount of labeled A that binds to the antibody. It is to be understoodthat specificity need not be absolute. For example, it is well known inthe art that numerous antibodies cross-react with other epitopes inaddition to those present in the target molecule. Such cross-reactivitymay be acceptable depending upon the application for which the antibodyis to be used. One of ordinary skill in the art will be able to selectantibodies having a sufficient degree of specificity to performappropriately in any given application (e.g., for detection of a targetmolecule, for therapeutic purposes, etc.). Specificity may be evaluatedin the context of additional factors such as the affinity of the bindingmolecule for the target molecule versus the affinity of the bindingmolecule for other targets (e.g., competitors). If a binding moleculeexhibits a high affinity for a target molecule that it is desired todetect and low affinity for non-target molecules, the antibody willlikely be an acceptable reagent for immunodiagnostic purposes. Once thespecificity of a binding molecule is established in one or morecontexts, it may be employed in other, preferably similar, contextswithout necessarily re-evaluating its specificity.

Stage of cancer: As used herein, the term “stage of cancer” refers to aqualitative or quantitative assessment of the level of advancement of acancer. Criteria used to determine the stage of a cancer include, butare not limited to, the size of the tumor and the extent of metastases(e.g., localized or distant).

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, or condition (cancer) has been diagnosed with and/or exhibitsone or more symptoms of the disease, disorder, or condition. In someembodiments, an individual who is suffering from cancer is an individualwho has increased tumor-associated or intratumoral cancer-relatedmarkers relative to an individual who does not have cancer.

Symptoms are reduced: According to the present invention, “symptoms arereduced” when one or more symptoms of a particular disease, disorder orcondition is reduced in magnitude (e.g., intensity, severity, etc.)and/or frequency. For purposes of clarity, a delay in the onset of aparticular symptom is considered one form of reducing the frequency ofthat symptom. Many cancer patients with smaller tumors have no symptoms.It is not intended that the present invention be limited only to caseswhere the symptoms are eliminated. The present invention specificallycontemplates treatment such that one or more symptoms is/are reduced(and the condition of the subject is thereby “improved”), albeit notcompletely eliminated.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that has a therapeutic effect and/or elicits a desiredbiological and/or pharmacological effect, when administered to asubject.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount of a therapeuticprotein which confers a therapeutic effect on the treated subject, at areasonable benefit/risk ratio applicable to any medical treatment. Thetherapeutic effect may be objective (i.e., measurable by some test ormarker) or subjective (i.e., subject gives an indication of or feels aneffect). In particular, the “therapeutically effective amount” refers toan amount of a therapeutic protein or composition effective to treat,ameliorate, or prevent a desired disease or condition, or to exhibit adetectable therapeutic or preventative effect, such as by amelioratingsymptoms associated with the disease, preventing or delaying the onsetof the disease, and/or also lessening the severity or frequency ofsymptoms of the disease. A therapeutically effective amount is commonlyadministered in a dosing regimen that may comprise multiple unit doses.For any particular therapeutic protein, a therapeutically effectiveamount (and/or an appropriate unit dose within an effective dosingregimen) may vary, for example, depending on route of administration, oncombination with other pharmaceutical agents. Also, the specifictherapeutically effective amount (and/or unit dose) for any particularpatient may depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific pharmaceutical agent employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and/orrate of excretion or metabolism of the specific fusion protein employed;the duration of the treatment; and like factors as is well known in themedical arts.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a substance that partiallyor completely alleviates, ameliorates, relives, inhibits, delays onsetof, reduces severity of, and/or reduces incidence of one or moresymptoms, features, and/or causes of a particular disease, disorder,and/or condition (e.g., cancer). Such treatment may be of a subject whodoes not exhibit signs of the relevant disease, disorder and/orcondition and/or of a subject who exhibits only early signs of thedisease, disorder, and/or condition. Alternatively or additionally, suchtreatment may be of a subject who exhibits one or more established signsof the relevant disease, disorder and/or condition. In some embodiments,treatment may be of a subject who has been diagnosed as suffering fromthe relevant disease, disorder, and/or condition. In some embodiments,treatment may be of a subject known to have one or more susceptibilityfactors that are statistically correlated with increased risk ofdevelopment of the relevant disease, disorder, and/or condition.

The terms PNA and PNA moiety are used interchangeably herein. The termsPNA agent and PNA derivative are used interchangeably herein.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention is based, in part, upon the discovery that it ispossible to produce peptide nucleic acid (PNA) agents that efficientlycross cell membranes and specifically target and suppress genes.Modifications to PNA agents improve the transmembrane permeability,target binding stability, solubility and specificity for target geneswith specific sequences.

In some embodiments, PNA agents comprising: a PNA-moiety; and a firstcationic and/or hydrophobic moiety at a first end of the PNA moiety; anda second cationic and/or hydrophobic moiety at a second end of the PNAmoiety are provided. In some embodiments, PNA agents wherein the firstand/or second cationic and/or hydrophobic moiety is or comprises apeptide are provided. In some embodiments, the first and/or secondcationic and/or hydrophobic peptide comprises or consists of lysineresidues. In some embodiments, at least one lysine residue comprises apalmitoyl side chain moiety. In some embodiments, the first and/orsecond cationic and/or hydrophobic peptide comprises amines. In someembodiments, PNA agents have a sequence that does not form hairpinloops. In some embodiments, PNA agents should have a sequence that doesnot form hairpin loops. In some embodiments, PNA agents should have asequence that contains less than 60% purines.

In some embodiments, the first and/or second cationic and/or hydrophobicmoiety is a targeting and delivery moiety that aids delivery of PNAthrough the cell membrane whereas the cationic moiety guides stabilityof the PNA against the target phosphate backbone. In some embodiments,the second cationic and/or hydrophobic moiety is or comprises a secondcationic peptide which functions in the same capacity as the firstcationic/hydrophobic moiety. In some embodiments, the cationic and/orhydrophobic moiety is at the PNA agent's N-terminus. In someembodiments, the targeting, or hydrophobic and/or cationic moiety is atthe PNA agent's C-terminus. In some embodiments, the first cationicand/or hydrophobic moiety is a targeting moiety that aids delivery ofPNA through the cell membrane and is attached at the PNA agent'sN-terminus; and the second cationic and/or hydrophobic moiety is orcomprises a cationic peptide that is attached at the PNA agent'sC-terminus and guides stability of the PNA against the target phosphatebackbone. In some embodiments, a second cationic peptide N-terminal tothe PNA peptide and C-terminal to the targeting moiety is provided.

In some embodiments, PNA agents having a sequence that targets a geneare provided. In some embodiments, PNA agents have a sequence thattargets a 13-20 nucleotide sequence of a gene with 75% or greatercomplementarity. In some embodiments, PNA agents have a sequence thattargets a nucleic acid whose length is at least 14, 15, 16, 17, or 18nucleotides and/or the complementarity is at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100%.

In some embodiments, the PNA component incorporates a sense (mRNA)sequence of a target gene.

In some embodiments, PNA agents have a sequence that targets anon-promoter region of a gene. In some embodiments, the gene is anoncogene. In some embodiments, oncogenes include a mutant sequenceelement and PNA agents have a sequence that targets a site comprising orconsisting of the mutant sequence element.

In some embodiments, PNA agents target a site comprising or consistingof a region of a BRAF oncogene comprising a mutation corresponding to aV600E mutation in a BRAF protein. In some embodiments, PNA agentstargets a site comprising or consisting of a region of a Gnaq genecomprising a mutation corresponding to a Q209L mutation in a Gnaqprotein.

In some embodiments, PNA agents target a site comprising or consistingof a region comprising a translocation junction of an oncogene. In someembodiments, PNA agents target a site comprising or consisting of aregion of a MYB-NFIB translocation comprising a junction of MYB and NFIBgenes or fragments thereof. In some embodiments, PNA agents target asite comprising or consisting of a region of a FUS-CHOP translocationcomprising a junction of FUS and CHOP genes or fragments thereof. Insome embodiments, PNA agents target a site comprising or consisting of aregion of a EWS-FLI1 translocation comprising a junction of EWS and FLI1genes or fragments thereof. In some embodiments, PNA agents target asite comprising or consisting of a region of a BCR-ABL translocationcomprising a junction of BCR and ABL genes or fragments thereof. In someembodiments, PNA agents target a site comprising or consisting of aregion of a SYT-SSX translocation comprising a junction of SYT and SSXgenes or fragments thereof. In some embodiments, PNA agents target atranslocation site comprising a juxtaposition/junction of two generegions or a gene region which contains a point mutation or multiplepoint mutations.

In some embodiments, PNA agents target a site comprising or consistingof a region comprising an amplification of a gene. In some embodiments,PNA agents target gene amplifications comprising AKT2, CDK4, MDM2, MYCN,CCNE, CCND1, KRAS, HRAS, EGFR, ERBB2, ERBB1, FGF, FGFR1, FGFR2, MYC,MYB, and MET.

In some embodiments, PNA agents have a sequence that targets a site in agene, which PNA agents are characterized in that, when a systemcomprising a cell that expresses the gene is exposed to the PNA agent,expression of the gene is reduced by an amount within the range of 50%to 100% when the PNA agent is present as compared with otherwisecomparable conditions when it is absent. In some embodiments, the idealrange is dependent upon the response measured by decreased cellproliferation. In some embodiments, the cell is a human cell. In someembodiments, the system is or comprises an animal. In some embodiments,the system is or comprises a primate. In some embodiments, the system isor comprises a human. In some embodiments, the system is or comprises aBRAF mouse. In some embodiments, the system is or comprises the cell inculture.

In some embodiments, PNA moieties have a length within the range of13-18 nucleotides. In some embodiments, PNA moieties have palmitoyllysine attached to the termini. In some embodiments, PNA moieties havepalmitoyl lysine attached to both N- and C-termini. In some embodiments,PNA moieties have a delivery peptide length within the range of 8-12amino acids. In some embodiments, PNA-peptide conjugates areintramolecularly stabilized by pi-interacting nucleoside bases. In someembodiments, the radius of gyration of the PNA agent is decreased withinthe range of 25% to 50% by hydrophobic solvent exclusion driving the N-and C-terminal palmitoyl lysines proximal to each other. In someembodiments, PNA agents, when contacted with a cell membrane, crossesthe membrane 10 times more quickly in comparison to reference PNA agentslacking one or both terminal hydrophobic/cationic moieties. In someembodiments, PNA agents cross membranes a magnitude more easily basedupon results from cell proliferation experiments comparing with andwithout terminal palmitoyl lysines.

In some embodiments, a PNA moiety has a first cationic moiety and afirst hydrophobic moiety at a first end, and a second cationic moietyand a second hydrophobic moiety at a second end, and the first cationicmoiety and the first hydrophobic moiety at the first end are part of oneamino acid, and the second cationic moiety and the second hydrophobicmoiety at the second end are part of one amino acid.

In some embodiments, methods for treating or reducing the risk of adisease, disorder, or condition comprising: administering to a subjectsusceptible to the disease, disorder, or condition PNA agents areprovided. In some embodiments, subjects suffering from or susceptible tocancer are provided. In some embodiments, cancers are selected frommelanoma, ocular melanoma and/or sarcoma.

In some embodiments, methods of reducing expression of a target gene ina cell comprising: contacting a cell in which the target is expressedwith at least one PNA agent; determining a level or activity of thetarget in the cell when the PNA agent is present as compared with atarget reference level or activity observed under otherwise comparableconditions when it is absent; and classifying the at least one PNA agentas a target inhibitor if the level or activity of the target issignificantly reduced when the PNA agent is present as compared with thetarget reference level or activity are provided.

In some embodiments, methods for identifying and/or characterizing PNAagents for target inhibition comprising: contacting a system in which atarget is expressed with at least one PNA agent; determining a level oractivity of the target in the system when the PNA agent is present ascompared with a target reference level or activity observed underotherwise comparable conditions when it is absent; and classifying theat least one PNA agent as a target inhibitor if the level or activity ofthe target is significantly reduced when the PNA agent is present ascompared with the target reference level or activity are provided. Insome embodiments, suppressing a tumor-driving oncogene is measured bydetermining the activity level by the amount of suppression of cellproliferation; the suppression of oncogene mRNA; and the suppression ofoncogene protein product. PNAs without any terminal (d)lysine-(d)lysinepalmitoyl lysine, with one terminal (d)lysine-(d)lysine-palmitoyllysine, and with both terminal (d)lysine-(d)lysine-palmitoyl lysine havebeen evaluated. In some embodiments, PNAs conjugated to NLS, TAT, or anyother delivery peptide, incorporating both terminal(d)lysine-(d)lysine-palmitoyl lysine show significantly better genesuppression than those with only a single terminus derivatized or notermini derivatized.

In some embodiments, one or more of the following PNA agents may be usedin connection with the present invention: PNAs with terminal peptidesincluding tetra-substituted ammonium or tri-substituted sulfoniummoieties.

In some embodiments, the level or activity of the target comprises atarget mRNA level. In some embodiments, the level or activity of thetarget comprises a target protein level. In some embodiments, the levelor activity of the target corresponds to cell viability. In someembodiments, a significant reduction in the level or activity of thetarget corresponds to a greater than 50% increase in cell viability. Insome embodiments, complete suppression of gene expression is notnecessary for significant suppression of cell proliferation/decreasingcell viability.

In some embodiments, the system comprises an in vitro system. In someembodiments, the system comprises an in vivo system. In someembodiments, the system is or comprises cells.

In some embodiments, the cells comprise cancer cells. In someembodiments, the system comprises cells in cell culture. In someembodiments, the cells in cell culture comprise BRAF wild type cells. Insome embodiments, BRAF wild type cells comprise C918 cells. In someembodiments, the cells in cell culture comprise BRAF V600E melanomacells. In some embodiments, BRAF V600E melanoma cells are selected fromOCM1A uveal melanoma cells and/or SK-MEL 7 cutaneous melanoma cells.

In some embodiments, the system is or comprises tissue. In someembodiments, the system is or comprises an organism. In someembodiments, the level or activity of the target corresponds to survivalof the organism. In some embodiments, a significant reduction in thelevel or activity of the target comprises a greater than 50% increase insurvival of the organism. In some embodiments, the organism comprises amouse. In some embodiments, the mouse comprises a BRAF mouse.

In some embodiments, a significant reduction in the level or activity ofthe target comprises a greater than 30-50% reduction of target activity.In some embodiments, a significant reduction in the level or activity ofthe target comprises a greater than 50-100% reduction of targetactivity. In some embodiments, a significant reduction in the level oractivity of the target comprises a greater than 50% reduction of targetlevels. In some embodiments, reduction of gene target expression by30-50% significantly reduces tumor cell proliferation in cell culture.

In some embodiments, the reference level is a historical reference. Insome embodiments, the historical reference is recorded in a tangibleand/or computer-readable medium.

In some embodiments, the target is a region comprising a point mutationin an oncogene. In some embodiments, the target is a region of a BRAFoncogene comprising a mutation corresponding to a V600E mutation in aBRAF protein. In some embodiments, the target is a region of a Gnaq genecomprising a mutation corresponding to a Q209L mutation in a Gnaqprotein. In some embodiments, the target is a region comprising atranslocation junction of an oncogene. In some embodiments, the targetis a MYB-NFIB translocation comprising a junction of MYB and NFIB genesor fragments thereof. In some embodiments, the target is a FUS-CHOPtranslocation comprising a junction of FUS and CHOP genes or fragmentsthereof. In some embodiments, the target is a BCR-ABL translocationcomprising a junction of BCR and ABL genes or fragments thereof. In someembodiments, the target is a SYT-SSX translocation comprising a junctionof SYT and SSX genes or fragments thereof. In some embodiments, thetarget is a region comprising a gene amplification. In some embodimentsthe gene amplification comprises an amplification of AKT2, CDK4, MDM2,MYCN, CCNE, CCND1, KRAS, HRAS, EGFR, ERBB2, ERBB1, FGF, FGFR1, FGFR2,MYC, MYB, and MET.

In some embodiments, a pharmaceutical composition comprising PNA agentsand pharmaceutically acceptable carriers are provided. In someembodiments, pharmaceutical composition is formulated for directadministration into a target tissue. In some embodiments, thepharmaceutical composition is formulated for oral administration. Insome embodiments, the pharmaceutical composition is formulated forparenteral administration. In some embodiments, the pharmaceuticalcomposition is formulated for intradermal administration. In someembodiments, the pharmaceutical composition is formulated fortransdermal administration. In some embodiments, the pharmaceuticalcomposition is formulated for administration by inhalation. In someembodiments, the pharmaceutical composition is or comprises a liquid. Insome embodiments, the pharmaceutical composition is or comprises asolid.

Peptide Nucleic Acid (PNA) Agent Structure

Peptide nucleic acids are synthetic polymers with similarities to DNAand RNA. PNAs possess backbones of repeating N-(2-aminoethyl)-glycineunits that are linked by peptide bonds. PNAs are also called PNAmoieties herein. This differs from backbones of DNA and RNA which arecomposed of deoxyribose and ribose sugar backbones, respectively.Furthermore, pyrimidine and purine bases are linked to the PNA backboneby carbonyl groups and methylene bridges. PNA backbones contain nocharged phosphate groups. Therefore, due to a lack of electrostaticrepulsion, binding between PNA sequences and DNA (or RNA) strands isstronger than binding between two DNA (or RNA) strands. Because of thehigher binding strength, PNA oligomers longer than 20-25 bases areusually not necessary. Increasing the length of PNA strands could reducespecificity for target DNA (or RNA) sequences. A PNA/DNA mismatch hasgreater instability than a DNA/DNA mismatch; PNAs exhibit greaterspecificity than DNA when binding to complementary sequences. The lackof charged phosphate groups also contributes to the hydrophobic natureof PNAs, which cannot cross cellular membranes without somemodification. These modifications can include, but are not limited to,covalently coupling a cell penetrating peptide and/or addingcationic/hydrophobic peptides. PNAs are also stable over a wide pH rangeand are resistant to enzyme degradation as they are not recognized byeither proteases or nucleases.

In some embodiments, PNA agents are complementary to a target sequence.In some embodiments, they are exact copies of a mRNA sequence expressedby a gene of interest. In some embodiments, this is also the sensestrand sequence of the gene. In some embodiments, PNA agents can becreated complementary to any gene of interest.

Embodiments of the present invention are drawn to methods of improvingthe ability of PNA agents to cross cellular membranes. In someembodiments, the physico-chemical properties of the PNA agents have beenmodified to improve delivery across cell membranes by addingcationic/hydrophobic delivery peptides. In some embodiments, thehydrophobic and cationic terminal peptides together facilitate passivetransport across membranes. PNA agents comprised of hydrophobice-palmitoyl lysines at the termini have improved capabilities forcrossing cellular membranes compared to standard PNA-peptide conjugates.In some embodiments, the terminal hydrophobic moieties in this designalso allow the termini to be hydrophobically driven together decreasingthe radius of gyration of the polymer. The smaller size allows forbetter transport. In some embodiments, having both ends of the PNApolymer derivatized more thoroughly imparts delivery functionalizationof this large molecule. Hydrophobic e-palmitoyl lysine termini aredriven together by solvent exclusion and the PNA-peptide conjugate isintramolecularly further stabilized by pi-interacting nucleoside bases.The PNA-peptide conjugate becomes more compact due to a decreased radiusof gyration, thus allowing it to more easily permeate lipid bilayers. Insome embodiments, the PNA agent is comprised of Lys(palmitoyl)-(dLys)2at the N- and C-termini, bracketing a delivery peptide of ˜10 aminoacids in length and a ˜15-18mer PNA. In some embodiments, the typicalstructure of PNA agents include: a delivery peptide of approximately 10amino acids in length and a ˜15-18mer PNA located within the bounds oftwo Lys(palmitoyl)-(dLys)2—termini attached by d-lysine. For example:

eg. Lys(palmitoyl)-(dLys)2-delivery peptide-PNA-(dLys)2—Lys(palmitoyl)

In some embodiments, PNA agents employ a modified NLS delivery peptide.In some embodiments, PNA agents employ a modified TAT delivery peptide.

In some embodiments, PNA agents used against a BRAF V600E targetinclude:

AcNH-Lys(palmitoyl)-dLys-dLys-C CTCAAGAGTAATAATAT-dLys-dPro-dLys-dLys-dLysdArg-dLys-dVal-dLys-dLys-Lys(palmitoyl)-CONH2[I-292-3 L2LP (employing a NLS delivery peptide)]

and

AcNH-Lys(palmitoyl)-dLys-dLys-CCTCAAGAGTAATAATAT-dLys-dArg3-dGln-dArg2-dLys2-dArg-Gly-dTyr-dLys-dLys-Lys(palmitoyl)-CONH2.[I-292-9 L2 (employing a modified TAT delivery peptide)]

Gene Targeting

In some embodiments, the cationically charged termini improve theability of the PNA to target genes with specific nucleic acid sequences.Stabilizing the cationically charged lysine-derivatized termini againstthe anionic DNA offers a kinetically faster binding by terminalnucleation as per the Zimm-Bragg statistical model. This enables the PNAto target non-promoter sequences in an improved manner, which isespecially unexpected given that promoter sequences are usuallyopen/unravelled and awaiting binding while non-promoter regions of genesare less accessible. PNA is stabilized against its DNA target merely forlacking repulsive anionic phosphate-phosphate repulsive forces(enthalpic advantage). The cationic ends of the PNA-peptide improve theentropic component of binding by stabilizing the more configurationallyfree termini of the PNA-peptide against the target.

In some embodiments, the PNA of the PNA-peptide conjugate is of standarddesign—the length range is usually from 13-18 bases. For lengths lessthan 13 bases the binding becomes much less thermodynamically favorabledue to decreased enthalpy of binding. For lengths greater than 18 basesdo not offer more of a thermodynamic advantage as the gain in enthalpicbinding energy is offset by the kinetic disadvantage of properlypositioning such a long strand (more intramolecular substrates couldcompete with the binding state).

In some embodiments, the PNA agents target specific genes or geneticsequences. PNA agents can be designed to target genes possessing knownmutated sequences as well as sites of genetic translocations. In someembodiments, the PNA agents target oncogenes. In some embodiments, thePNA agents can target mutant oncogenes. In some embodiments, wild typeand mutant oncogenes that can be targeted are selected from the groupcomprising ABL1, ABL2, AKT1, AKT2, ALK, ATF1, BCL11A, BCL2, BLC3, BCL6,BCR, BRAF, CARD11, CBLB, CBLC, CCND1, CCND2, CCND3, CDX2, CTNNB1, DDB2,DDIT3, DDX6, DEK, EGFR, ELK4, ERBB2, ETV4, ETV6, EVIL EWSR1, FEV, FGFR1,FGFR1OP, FGFR2, FUS, GOLGA5, HMGA1, HMGA2, HRAS, IDHL IDH2, IRF4, JUN,KIT, KRAS, LCK, LMO2, MAF, MAFB, MAML2, MDM2, MET, MITF, MLL, MPL, MYB,MYC, MYCL1, MYCN, NCOA4, NFKB2, NRAS, NTRK1, NUP214, PAX8, PDGFB,PIK3CA, PIM1, PLAG1, PPARG, PTPN11, RAFT, REL, RET, ROS1, SMO, SS18,TCL1A, TET2, TFG, TLX1, TPR, and USP6. In some embodiments, theoncogenes targeted comprise BRAF and Gnaq. In some embodiments, PNAagents can target sites of genetic abnormalities. In some embodiments,PNA agents can target sites of translocations. In some embodiments,sites of translocation can comprise the junction of the MYB-NFIBtranslocation. In some embodiments, sites of translocation can comprisethe junction of the FUS-CHOP translocation. In some embodiments, sitesof translocation can comprise the junction of the BCR-ABL translocation.In some embodiments, sites of translocation can comprise the junction ofthe SYT-SSX translocation. Any gene sequence can be targeted;PNA-peptide agents presented are destined for targeting againstoncogenes comprised of single point mutations (and/or combinationsthereof), gene translocation points, gene amplifications, or overexpressed wild-type genes that drive tumors.

Systems for Testing PNA Agents

PNA agents can be tested in cancer lines as well as cell linesexpressing genetic abnormalities. In some embodiments, PNA agents aretested in cancer cell lines. In some embodiments, PNA agents are testedin melanoma cell lines. In some embodiments, PNA agents are tested inuveal and cutaneous melanoma, and Ewings sarcoma cell lines. PNA agentscan be tested in cell lines expressing a genetic abnormality associatedwith a disease other than cancer. PNA agents can be tested in neuronaland/or muscle cell lines with aberrant gene expression. In someembodiments, PNA agents are tested in rodent models comprising mutantgene sequences. In some embodiments, PNA agents may be tested in anycell line. A variety of cell lines are employed for testing PNA peptidesby one of ordinary skill in the art.

Applications of PNA Agents

Targeting and binding by PNA agents would have uses as research tools,medical diagnostics and pharmaceutical treatments. In some embodiments,PNA agents can be used to target and bind specific genetic sequences. Insome embodiments, PNA agents can be used to suppress expression ofgenetic sequences. PNA agents targeted to specific genes can serve asvaluable research tools in understanding the function of those genes.Suppressing the expression of particular gene products would helpelucidate and discover the role of those products in differentbiological pathways.

In some embodiments, PNA agents are used to target and suppressexpression of BRAF genes. In some embodiments, PNA agents are used totarget and suppress expression of mutant BRAF genes. In someembodiments, PNA agents are used to target and suppress expression ofmutant Gnaq genes. In some embodiments, PNA agents can be used a treatdiseases associated with genetic sequences. In some embodiments, PNAagents are used to treat diseases associated with mutated BRAF genes. Insome embodiments, PNA agents are used to treat diseases associate withmutated Gnaq genes. In some embodiments, PNA agents are used to treatcancer.

In some embodiments, PNA agents are used to treat cancer in animals. Insome embodiments, PNA agents are used to treat cancer in mammals. Insome embodiments, PNA agents are used to treat cancer in primates. Insome embodiments, PNA agents are used to treat cancer in humans.

PNA agents can target and bind to mutated genetic sequences and suppressthe expression of mutant oncogenes, thereby suppressing and/or treatingcancer. In some embodiments, PNA agents are used to treat cancer due tomutated BRAF genes. In some embodiments, PNA agents are used to treatcancer due to mutated Gnaq genes. In some embodiments, PNA agents areused to treat cancer due to translocations. PNA agents can target andbind to junctions of genetic translocations and suppress the expressionof mutated genetic sequences, thereby preventing and/or treatingtranslocation-associated cancer. Junctions of the MYB-NFIB translocationand the FUS-CHOP translocation can be targeted and suppressed by PNAagents of the present disclosure. Junctions of the BCR-ABL translocationand the SYT-SSX translocation can be targeted and suppressed by PNAagents of the present disclosure. PNA agents can target and bind tojunctions of gene amplifications and suppress the expression of mutatedgenetic sequences, thereby preventing and/or treatingamplification-associated cancer. Gene amplifications comprising thegenes AKT2, CDK4, MDM2, MYCN, CCNE, CCND1, KRAS, HRAS, EGFR, ERBB2,ERBB1, FGF, FGFR1, FGFR2, MYC, MYB, and MET can be targeted andsuppressed by PNA agents of the present disclosure.

PNA agents can be used to treat genetic abnormalities in diseases notassociated with cancer. Diseases or conditions caused by a mutated geneproduct can treated by targeting a PNA agent to the mutated geneexpressing the harmful gene product. Inherited or inborn disorders canbe treated through the use of targeted PNA agents. PNA agents can beused to suppress normal genes such as those supportive of obesity,metabolic syndromes, or related vasculopathies. PNA agents can be usedto target fungal genes, viral genes or bacterial genes causinginfection.

Pharmaceutical Compositions

The present invention also provides compositions comprising one or moreprovided antibodies, fragments or characteristic portions thereof. Insome embodiments, the present invention provides at least onePNA-conjugate and at least one pharmaceutically acceptable excipient.Such pharmaceutical compositions may optionally comprise and/or beadministered in combination with one or more additional therapeuticallyor biologically active substances. In some embodiments, providedpharmaceutical compositions are useful in medicine or the manufacture ofmedicaments. In some embodiments, provided pharmaceutical compositionsare useful as prophylactic agents (i.e., vaccines) in the treatment orprevention of cancer and neurodegenerative disorders thereof. In someembodiments, provided pharmaceutical compositions are useful intherapeutic applications, for example in individuals suffering fromcancer; e.g., as delivery vehicles capable of specifically targetingcytotoxic agents or compounds that block aberrant cellular signaling. Insome embodiments, the pharmaceutical compositions are simultaneouslyuseful in diagnostic applications and therapeutic applications. In someembodiments, pharmaceutical compositions are formulated foradministration to humans. In some embodiments, the pharmaceuticalcompositions comprise an antibody in combination with or conjugated to atherapeutic agent or other therapeutic as defined herein.

For example, pharmaceutical compositions may be provided in a sterileinjectable form (e.g., a form that is suitable for subcutaneousinjection or intravenous infusion). In some embodiments, pharmaceuticalcompositions are provided in a liquid dosage form that is suitable forinjection. In some embodiments, pharmaceutical compositions are providedas powders (e.g., lyophilized and/or sterilized), optionally undervacuum, which are reconstituted with an aqueous diluent (e.g., water,buffer, salt solution, etc.) prior to injection. In some embodiments,pharmaceutical compositions are diluted and/or reconstituted in water,sodium chloride solution, sodium acetate solution, benzyl alcoholsolution, phosphate buffered saline, etc. In some embodiments, powdershould be mixed gently with the aqueous diluent (e.g., not shaken).

In some embodiments, provided pharmaceutical compositions comprise oneor more pharmaceutically acceptable excipients (e.g., preservative,inert diluent, dispersing agent, surface active agent and/or emulsifier,buffering agent, etc.). In some embodiments, pharmaceutical compositionscomprise one or more preservatives. In some embodiments, pharmaceuticalcompositions comprise no preservatives.

In some embodiments, pharmaceutical compositions are provided in a formthat can be refrigerated and/or frozen. In some embodiments,pharmaceutical compositions are provided in a form that cannot berefrigerated and/or frozen. In some embodiments, reconstituted solutionsand/or liquid dosage forms may be stored for a certain period of timeafter reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days,7 days, 10 days, 2 weeks, a month, two months, or longer). In someembodiments, storage of antibody compositions for longer than thespecified time results in antibody degradation.

Liquid dosage forms and/or reconstituted solutions may compriseparticulate matter and/or discoloration prior to administration. In someembodiments, a solution should not be used if discolored or cloudyand/or if particulate matter remains after filtration.

Pharmaceutical compositions described herein may be prepared by anymethod known or hereafter developed in the art of pharmacology. In someembodiments, such preparatory methods include the step of bringingactive ingredient into association with one or more excipients and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient; for example, a peptidenucleic acid agent. The amount of the active ingredient is generallyequal to a dose that would be administered to a subject and/or aconvenient fraction of such a dose such as, for example, one-half orone-third of such a dose.

Relative amounts of active ingredient, pharmaceutically acceptableexcipient, and/or any additional ingredients in a pharmaceuticalcomposition in accordance with the invention may vary, depending uponthe identity, size, and/or condition of the subject treated and/ordepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

Pharmaceutical compositions of the present invention may additionallycomprise a pharmaceutically acceptable excipient, which, as used herein,may be or comprise solvents, dispersion media, diluents, or other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21st Edition,A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md., 2006)discloses various excipients used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional excipient medium is incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention.

Conjugates Generally

Multifunctional agents described herein comprise multiple entities, eachhaving at least one function. Certain embodiments of contemplatedmultifunctional agents comprise a targeting entity and at least one ofthe following entities: a detection entity, a therapeutic entity, and adiagnostic entity. In some embodiments, a multifunctional agent of theinvention contains a targeting entity, a therapeutic entity and adetection entity. In some embodiments, the entities of an agent may beconjugated to one another. Conjugation of various entities to form amultifunctional agent is not limited to particular modes of conjugation.For example, two entities may be covalently conjugated directly to eachother. Alternatively, two entities may be indirectly conjugated to eachother, such as via a linker entity. In some embodiments, amultifunctional agent may include different types of conjugation withinthe agent, such that some entities of the agent are conjugated viadirect conjugation while other entities of the agent are indirectlyconjugated via one or more linkers. In some embodiments, amultifunctional agent of the invention comprises a single type of alinker entity. In some embodiments, a multifunctional agent of theinvention comprises more than one type of linker entities. In someembodiments, a multifunctional agent includes a single type of linkerentities but of varying length.

In some embodiments, there is a covalent association between or amongentities contained in a multifunctional agent. As will be appreciated byone skilled in the art, the moieties may be attached to each othereither directly or indirectly (e.g., through a linker, as describedbelow).

In some embodiments, where one entity (such as a targeting entity) and asecond entity of a multifunctional agent are directly covalently linkedto each other, such direct covalent conjugation can be through a linkage(e.g., a linker or linking entity) such as an amide, ester,carbon-carbon, disulfide, carbamate, ether, thioether, urea, thiourea,isothiourea, amine, or carbonate linkage. Covalent conjugation can beachieved by taking advantage of functional groups present on the firstentity and/or the second entity of the multifunctional agent.Alternatively, a non-critical amino acid may be replaced by anotheramino acid that will introduce a useful group (such as amino, carboxy orsulfhydryl) for coupling purposes. Alternatively, an additional aminoacid may be added to at least one of the entities of the multifunctionalagent to introduce a useful group (such as amino, carboxy or sulfhydryl)for coupling purposes. Suitable functional groups that can be used toattach moieties together include, but are not limited to, amines,anhydrides, hydroxyl groups, carboxy groups, thiols, and the like. Anactivating agent, such as a carbodiimide, can be used to form a directlinkage. A wide variety of activating agents are known in the art andare suitable for conjugating one entity to a second entity.

In some embodiments, entities of a multifunctional agent embraced by thepresent invention are indirectly covalently linked to each other via alinker group. Such a linker group may also be referred to as a linker ora linking entity. This can be accomplished by using any number of stablebifunctional agents well known in the art, including homofunctional andheterofunctional agents (for examples of such agents, see, e.g., PierceCatalog and Handbook). The use of a bifunctional linker differs from theuse of an activating agent in that the former results in a linkingmoiety being present in the resulting conjugate (agent), whereas thelatter results in a direct coupling between the two moieties involved inthe reaction. The role of a bifunctional linker may be to allow reactionbetween two otherwise inert moieties. Alternatively or additionally, thebifunctional linker that becomes part of the reaction product may beselected such that it confers some degree of conformational flexibilityto the agent (e.g., the bifunctional linker comprises a straight alkylchain containing several atoms, for example, the straight alkyl chaincontains between 2 and 10 carbon atoms). Alternatively or additionally,the bifunctional linker may be selected such that the linkage formedbetween a provided antibody and therapeutic agent is cleavable, e.g.,hydrolysable (for examples of such linkers, see e.g. U.S. Pat. Nos.5,773,001; 5,739,116 and 5,877,296, each of which is incorporated hereinby reference in its entirety). Such linkers, for example, may be usedwhen higher activity of certain entities, such as a targeting agentand/or of a therapeutic entity is observed after hydrolysis of theconjugate. Exemplary mechanisms by which an entity may be cleaved from amultifunctional agent include hydrolysis in the acidic pH of thelysosomes (hydrazones, acetals, and cis-aconitate-like amides), peptidecleavage by lysosomal enzymes (the capthepsins and other lysosomalenzymes), and reduction of disulfides). Another mechanism by which suchan entity is cleaved from the multifunctional agent includes hydrolysisat physiological pH extra- or intra-cellularly. This mechanism applieswhen the crosslinker used to couple one entity to another entity is abiodegradable/bioerodible component, such as polydextran and the like.

For example, hydrazone-containing multifunctional agents can be madewith introduced carbonyl groups that provide the desired releaseproperties. Multifunctional agents can also be made with a linker thatcomprises an alkyl chain with a disulfide group at one end and ahydrazine derivative at the other end. Linkers containing functionalgroups other than hydrazones also have the potential to be cleaved inthe acidic milieu of lysosomes. For example, multifunctional agents canbe made from thiol-reactive linkers that contain a group other than ahydrazone that is cleavable intracellularly, such as esters, amides, andacetals/ketals.

Another example of class of pH sensitive linkers are the cis-aconitates,which have a carboxylic acid group juxtaposed to an amide group. Thecarboxylic acid accelerates amide hydrolysis in the acidic lysosomes.Linkers that achieve a similar type of hydrolysis rate acceleration withseveral other types of structures can also be used.

Another potential release method for conjugates of the therapeuticagents is the enzymatic hydrolysis of peptides by the lysosomal enzymes.In one example, a provided antibody is attached via an amide bond topara-aminobenzyl alcohol and then a carbamate or carbonate is madebetween the benzyl alcohol and the therapeutic agent. Cleavage of thepeptide leads to collapse of the amino benzyl carbamate or carbonate,and release of the therapeutic agent. In another example, a phenol canbe cleaved by collapse of the linker instead of the carbamate. Inanother variation, disulfide reduction is used to initiate the collapseof a para-mercaptobenzyl carbamate or carbonate.

Useful linkers which may be used as a linking entity of amultifunctional agent provided herein include, without limitation:polyethylene glycol, a copolymer of ethylene glycol, a polypropyleneglycol, a copolymer of propylene glycol, a carboxymethylcellulose, apolyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, anethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinylpyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer,a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylatedpolyol, a polyvinyl alcohol, a linear or branched glycosylated chain, apolyacetal, a long chain fatty acid, a long chain hydrophobic aliphaticgroup.

Some embodiments of the invention utilize multifunctional agents thatinclude at least one non-covalently associated entity. Examples ofnon-covalent interactions include, but are not limited to, hydrophobicinteractions, electrostatic interactions, dipole interactions, van derWaals interactions, and hydrogen bonding. Irrespective of the nature ofthe binding, interaction, or coupling, the association between a firstentity and a second entity is, in some embodiments, selective, specificand strong enough so that the second entity contained in the agent doesnot dissociate from the first entity before or during transport/deliveryto and into the target. Thus, association among multiple entities of amultifunctional agent may be achieved using any chemical, biochemical,enzymatic, or genetic coupling known to one skilled in the art.

Therapeutic Conjugates

As described herein, PNA agents may comprise part of multifunctionalagents with therapeutic utility related to cancer or neurodegenerativedisorders. Examples of therapeutic utilities in the context of thepresent disclosure include, without limitation, utility associated withtargeting (e.g., binding specific gene sequences), utility associatedwith therapeutic effects (e.g., cytotoxic and/or cytostatic effects,anti-proliferative effects, anti-angiogenic effects, reducing symptomsetc.), and utility associated with diagnosis, detection or labeling,etc.

A targeting entity is a molecular structure that can be contained in anagent which affects or controls the site of action by specificallyinteracting with, or has affinity for, a target of interest. As anexample, a target may be a molecule or molecular complex present on acell surface, e.g., certain cell types, tissues, etc. In someembodiments of the invention, the target is tumor-associated orintratumoral gene and the targeting entity is a PNA agent. Use oftargeting moieties for agents such as therapeutic agents is known in theart. In the context of the present application, primary or metastaticcancer cells, as well as other cell types, are the target. That is, atthe molecular level, a target is a molecule or cellular constituent thatis present (e.g., preferentially expressed) on a cell, such that it canspecifically or preferentially bind to PNA agents upon contact. The PNAagents of the invention exert specificity for their target (e.g.,oncogenes of cancer cells) and are able to localize to nuclei and bindto their target. In some embodiments, PNA agent targeting entitieslocalize to cancer cells and retain their association over a period oftime. In some embodiments, the PNA agent targets are the nucleic acidsequences encoding intratumoral and/or integral membrane proteins.

In some embodiments, the PNA agents are multifunctional agentscomprising a gene targeting entity, which essentially consists of a PNAagent, conjugated to one or more therapeutic agents. Non-limitingembodiments of useful conjugates of PNA agents that may be used in thediagnosis or assessment of, treatment of and the manufacture ofmedicaments for cancer or other disorders are provided below.

Nucleic acid anti-cancer agents suitable for use in the practice of thepresent invention include those agents that target genes associated withtumorigenesis and cell growth or cell transformation (e.g.,proto-oncogenes, which code for proteins that stimulate cell division),angiogenic/anti-angiogenic genes, tumor suppressor genes (which code forproteins that suppress cell division), genes encoding proteinsassociated with tumor growth and/or tumor migration, and suicide genes(which induce apoptosis or other forms of cell death), especiallysuicide genes that are most active in rapidly dividing cells.

Examples of genes associated with tumorigenesis and/or celltransformation include MLL fusion genes, BCR-ABL, TEL-AML1, EWS-FLI1,TLS-FUS, PAX3-FKHR, Bcl-2, AML1-ETO, AML1-MTG8, Ras, Fos PDGF, RET, APC,NF-1, Rb, p53, MDM2 and the like; overexpressed genes such as multidrugresistance genes; cyclins; beta-Catenin; telomerase genes; c-myc, n-myc,Bcl-2, Erb-B1 and Erb-B2; and mutated genes such as Ras, Mos, Raf, andMet. Examples of tumor suppressor genes include, but are not limited to,p53, p21, RB1, WT1, NF1, VHL, APC, DAP kinase, p16, ARF, Neurofibromin,and PTEN. Examples of genes that can be targeted by nucleic acid agentsuseful in anti-cancer therapy include genes encoding proteins associatedwith tumor migration such as integrins, selectins, andmetalloproteinases; anti-angiogenic genes encoding proteins that promoteformation of new vessels such as Vascular Endothelial Growth Factor(VEGF) or VEGFr; anti-angiogenic genes encoding proteins that inhibitneovascularization such as endostatin, angiostatin, and VEGF-R2; andgenes encoding proteins such as interleukins, interferon, fibroblastgrowth factor (α-FGF and (β-FGF), insulin-like growth factor (e.g.,IGF-1 and IGF-2), Platelet-derived growth factor (PDGF), tumor necrosisfactor (TNF), Transforming Growth Factor (e.g., TGF-α and TGF-β,Epidermal growth factor (EGF), Keratinocyte Growth Factor (KGF), stemcell factor and its receptor c-Kit (SCF/c-Kit) ligand, CD40L/CD40, VLA-4VCAM-1, ICAM-1/LFA-1, hyalurin/CD44, and the like.

PNA agents may have any of a variety of uses including, for example, useas anti-cancer or other therapeutic agents, probes, primers, etc.Nucleic acid agents may have enzymatic activity (e.g., ribozymeactivity), gene expression inhibitory activity (e.g., as antisense orsiRNA agents, etc), and/or other activities. Nucleic acids agents may beactive themselves or may be vectors that deliver active nucleic acidagents (e.g., through replication and/or transcription of a deliverednucleic acid). For purposes of the present specification, such vectornucleic acids are considered “therapeutic agents” if they encode orotherwise deliver a therapeutically active agent, even if they do notthemselves have therapeutic activity.

In some embodiments, conjugates of PNA agents comprise a nucleic acidtherapeutic agent that is a ribozyme. As used herein, the term“ribozyme” refers to a catalytic RNA molecule that can cleave other RNAor DNA molecules in a target-specific manner. Ribozymes can be used todownregulate the expression of any undesirable products of genes ofinterest. Examples of ribozymes that can be used in the practice of thepresent invention include, but are not limited to, those specific foroncogene mRNA or DNA.

In some embodiments, entities or moieties within conjugates of the PNAagents comprise a photosensitizer used in photodynamic therapy (PDT). InPDT, local or systemic administration of a photosensitizer to a patientis followed by irradiation with light that is absorbed by thephotosensitizer in the tissue or organ to be treated. Light absorptionby the photosensitizer generates reactive species (e.g., radicals) thatare detrimental to cells. For maximal efficacy, a photosensitizertypically is in a form suitable for administration, and also in a formthat can readily undergo cellular internalization at the target site,often with some degree of selectivity over normal tissues.

Conjugates of PNA agents associated with a photosensitizer can be usedas new delivery systems in PDT. In addition to reducing photosensitizeraggregation, delivery of photosensitizers according to the presentinvention exhibits other advantages such as increased specificity fortarget tissues/organ and cellular internalization of thephotosensitizer.

Photosensitizers suitable for use in the present invention include anyof a variety of synthetic and naturally occurring molecules that havephotosensitizing properties useful in PDT. In some embodiments, theabsorption spectrum of the photosensitizer is in the visible range,typically between 350 nm and 1200 nm, preferably between 400 nm and 900nm, e.g., between 600 nm and 900 nm. Suitable photosensitizers that canbe coupled to toxins according to the present invention include, but arenot limited to, porphyrins and porphyrin derivatives (e.g., chlorins,bacteriochlorins, isobacteriochlorins, phthalocyanines, andnaphthalocyanines); metalloporphyrins, metallophthalocyanines,angelicins, chalcogenapyrrillium dyes, chlorophylls, coumarins, flavinsand related compounds such as alloxazine and riboflavin, fullerenes,pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540),pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes,phenothiaziniums, methylene blue derivatives, naphthalimides, nile bluederivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins,and cercosporins), psoralens, quinones, retinoids, rhodamines,thiophenes, verdins, xanthene dyes (e.g., eosins, erythrosins, rosebengals), dimeric and oligomeric forms of porphyrins, and prodrugs suchas 5-aminolevulinic acid (R. W. Redmond and J. N. Gamlin, Photochem.Photobiol., 1999, 70: 391-475).

Exemplary photosensitizers suitable for use in the present inventioninclude those described in U.S. Pat. Nos. 5,171,741; 5,171,749;5,173,504; 5,308,608; 5,405,957; 5,512,675; 5,726,304; 5,831,088;5,929,105; and 5,880,145 (the contents of each of which are incorporatedherein by reference in their entirety).

In some embodiments, conjugates of PNA agents comprise aradiosensitizer. As used herein, the term “radiosensitizer” refers to amolecule, compound or agent that makes tumor cells more sensitive toradiation therapy. Administration of a radiosensitizer to a patientreceiving radiation therapy generally results in enhancement of theeffects of radiation therapy. The advantage of coupling aradiosensitizer to a targeting entity (e.g., PNA agents capable oftargeting intratumoral genetic sequences) is that the radiosensitizeeffects only on target cells. For ease of use, a radiosensitizer shouldalso be able to find target cells even if it is administeredsystemically. However, currently available radiosensitizers aretypically not selective for tumors, and they are distributed bydiffusion in a mammalian body. PNA agents conjugates of the presentinvention can be used as a new delivery system for radiosensitizers.

A variety of radiosensitizers are known in the art. Examples ofradiosensitizers suitable for use in the present invention include, butare not limited to, paclitaxel (TAXOL®), carboplatin, cisplatin, andoxaliplatin (Amorino et al., Radiat. Oncol. Investig., 1999, 7: 343-352;Choy, Oncology, 1999, 13: 22-38; Safran et al., Cancer Invest., 2001,19: 1-7; Dionet et al., Anticancer Res., 2002, 22: 721-725; Cividalli etal., Radiat. Oncol. Biol. Phys., 2002, 52: 1092-1098); gemcitabine(Gemzar®) (Choy, Oncology, 2000, 14: 7-14; Mornex and Girard, Annals ofOncology, 2006, 17: 1743-1747); etanidazole (Nitrolmidazole®) (Inanamiet al., Int. J. Radiat. Biol., 2002, 78: 267-274); misonidazole(Tamulevicius et al., Br. J. Radiology, 1981, 54: 318-324; Palcic etal., Radiat. Res., 1984, 100: 340-347), tirapazamine (Masunaga et al.,Br. J. Radiol., 2006, 79: 991-998; Rischin et al., J. Clin. Oncol.,2001, 19: 535-542; Shulman et al., Int. J. Radiat. Oncol. Biol. Phys.,1999, 44: 349-353); and nucleic acid base derivatives, e.g., halogenatedpurines or pyrimidines, such as 5-fluorodeoxyuridine (Buchholz et al.,Int. J. Radiat. Oncol. Biol. Phys., 1995, 32: 1053-1058).

In some embodiments, conjugates of PNA agents comprise a radioisotope.Examples of suitable radioisotopes include any α-, β- or γ-emitter,which, when localized at a tumor site, results in cell destruction (S.E. Order, “Analysis, Results, and Future Prospective of the TherapeuticUse of Radiolabeled Antibody in Cancer Therapy”, Monoclonal Antibodiesfor Cancer Detection and Therapy, R. W. Baldwin et al. (Eds.), AcademicPress, 1985). Examples of such radioisotopes include, but are notlimited to, iodine-131 (¹³¹I), iodine-125 (¹²⁵I), bismuth-212 (²¹²Bi),bismuth-213 (²¹³Bi), astatine-211 (²¹¹At), rhenium-186 (¹⁸⁶Re),rhenium-188 (¹⁸⁸Re), phosphorus-32 (³²P), yttrium-90 (⁹⁰Y), samarium-153(¹⁵³Sm), and lutetium-177 (¹⁷⁷Lu).

In some embodiments, conjugates of the PNA agents may be used indirected enzyme prodrug therapy. In a directed enzyme prodrug therapyapproach, a directed/targeted enzyme and a prodrug are administered to asubject, wherein the targeted enzyme is specifically localized to aportion of the subject's body where it converts the prodrug into anactive drug. The prodrug can be converted to an active drug in one step(by the targeted enzyme) or in more than one step. For example, theprodrug can be converted to a precursor of an active drug by thetargeted enzyme. The precursor can then be converted into the activedrug by, for example, the catalytic activity of one or more additionaltargeted enzymes, one or more non-targeted enzymes administered to thesubject, one or more enzymes naturally present in the subject or at thetarget site in the subject (e.g., a protease, phosphatase, kinase orpolymerase), by an agent that is administered to the subject, and/or bya chemical process that is not enzymatically catalyzed (e.g., oxidation,hydrolysis, isomerization, epimerization, etc.).

Some embodiments of the invention utilize PNA agent-directed enzymeprodrug therapy, wherein a PNA agent is linked to an enzyme and injectedin a subject, resulting in selective binding of the enzyme totumor-associated or metastatic genes. Subsequently, a prodrug isadministered to the subject. The prodrug is converted to its active formby the enzyme only within or nearby the cancer cells. Selectivity isachieved by the specificity of the PNA agents and by delaying prodrugadministration until there is a large differential between cancer andnormal tissue enzyme levels. Cancer cells may also be targeted with thegenes encoding for prodrug activating enzymes. This approach has beencalled virus-directed enzyme prodrug therapy (VDEPT) or more generallyGDEPT (gene-directed enzyme prodrug therapy, and has shown good resultsin laboratory systems. Other versions of directed enzyme prodrug therapyinclude PDEPT (polymer-directed enzyme prodrug therapy), LEAPT(lectin-directed enzyme-activated prodrug therapy), and CDEPT(clostridial-directed enzyme prodrug therapy).

Nonlimiting examples of enzyme/prodrug/active drug combinations suitablefor use in the present invention are described, for example, in Bagshaweet al., Current Opinions in Immunology, 1999, 11: 579-583; Wilman,“Prodrugs in Cancer Therapy”, Biochemical Society Transactions, 14:375-382, 615th Meeting, Belfast, 1986; Stella et al., “Prodrugs: AChemical Approach To Targeted Drug Delivery”, in “Directed DrugDelivery”, Borchardt et al., (Eds), pp. 247-267 (Humana Press, 1985).Nonlimiting examples of enzyme/prodrug/active anti-cancer drugcombinations are described, for example, in Rooseboom et al., Pharmacol.Reviews, 2004, 56: 53-102.

Examples of prodrug activating enzymes include, but are not limited to,nitroreductase, cytochrome P450, purine-nucleoside phosphorylase,thymidine kinase, alkaline phosphatase, β-glucuronidase,carboxypeptidase, penicillin amidase, β-lactamase, cytosine deaminase,and methionine γ-lyase.

Examples of anti-cancer drugs that can be formed in vivo by activationof a prodrug by a prodrug activating enzyme include, but are not limitedto, 5-(aziridin-1-yl)-4-hydroxyl-amino-2-nitro-benzamide,isophosphoramide mustard, phosphoramide mustard, 2-fluoroadenine,6-methylpurine, ganciclovir-triphosphate nucleotide, etoposide,mitomycin C, p-[N,N-bis(2-chloroethyl)amino]phenol (POM), doxorubicin,oxazolidinone, 9-aminocamptothecin, mustard, methotrexate, benzoic acidmustard, adriamycin, daunomycin, carminomycin, bleomycins, esperamicins,melphalan, palytoxin, 4-desacetylvinblastine-3-carboxylic acidhydrazide, phenylenediamine mustard,4′-carboxyphthalato(1,2-cyclohexane-diamine) platinum, taxol,5-fluorouracil, methylselenol, and carbonothionic difluoride.

In some embodiments, a therapeutic (e.g., anti-cancer) agent comprises aconjugate of one or more PNA agents and an anti-angiogenic agent.Antiangiogenic agents suitable for use in the present invention includeany molecule, compound, or factor that blocks, inhibits, slows down, orreduces the process of angiogenesis, or the process by which new bloodvessels form by developing from preexisting vessels. Such a molecule,compound, or factor can block angiogenesis by blocking, inhibiting,slowing down, or reducing any of the steps involved in angiogenesis,including (but not limited to) steps of (1) dissolution of the membraneof the originating vessel, (2) migration and proliferation ofendothelial cells, and (3) formation of new vasculature by migratingcells.

Examples of anti-angiogenic agents include, but are not limited to,bevacizumab (AVASTIN®), celecoxib (CELEBREX®), endostatin, thalidomide,EMD121974 (Cilengitide), TNP-470, squalamine, combretastatin A4,interferon-α, anti-VEGF antibody, SU5416, SU6668, PTK787/2K 22584,Marimistal, AG3340, COL-3, Neovastat, and BMS-275291.

Administration

PNA agents in accordance with the invention and pharmaceuticalcompositions of the present invention may be administered according toany appropriate route and regimen. In some embodiments, a route orregimen is one that has been correlated with a positive therapeuticbenefit.

In some embodiments, the exact amount administered may vary from subjectto subject, depending on one or more factors as is well known in themedical arts. Such factors may include, for example, one or more ofspecies, age, general condition of the subject, the particularcomposition to be administered, its mode of administration, its mode ofactivity, the the severity of disease; the activity of the specific PNAagents employed; the specific pharmaceutical composition administered;the half-life of the composition after administration; the age, bodyweight, general health, sex, and diet of the subject; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed and thelike. Pharmaceutical compositions may be formulated in dosage unit formfor ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by an attending physician withinthe scope of sound medical judgment.

Compositions of the present invention may be administered by any route,as will be appreciated by those skilled in the art. In some embodiments,compositions of the present invention are administered by oral (PO),intravenous (IV), intramuscular (IM), intra-arterial, intramedullary,intrathecal, subcutaneous (SQ), intraventricular, transdermal,interdermal, intradermal, rectal (PR), vaginal, intraperitoneal (IP),intragastric (IG), topical (e.g., by powders, ointments, creams, gels,lotions, and/or drops), mucosal, intranasal, buccal, enteral, vitreal,sublingual; by intratracheal instillation, bronchial instillation,and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/orthrough a portal vein catheter.

In some embodiments, PNA agents in accordance with the present inventionand/or pharmaceutical compositions thereof may be administeredintravenously, for example, by intravenous infusion. In someembodiments, PNA agents in accordance with the present invention and/orpharmaceutical compositions thereof may be administered by intramuscularinjection. In some embodiments, PNA agents in accordance with thepresent invention and/or pharmaceutical compositions thereof may beadministered by intratumoural injection. In some embodiments, PNA agentsin accordance with the present invention and/or pharmaceuticalcompositions thereof may be administered by subcutaneous injection. Insome embodiments, PNA agents in accordance with the present inventionand/or pharmaceutical compositions thereof may be administered viaportal vein catheter. However, the invention encompasses the delivery ofPNA agents in accordance with the present invention and/orpharmaceutical compositions thereof by any appropriate route taking intoconsideration likely advances in the sciences of drug delivery.

In some embodiments, PNA agents in accordance with the present inventionand/or pharmaceutical compositions thereof may be administered at dosagelevels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg,from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kgto about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about1 mg/kg to about 25 mg/kg of subject body weight per day to obtain thedesired therapeutic effect. The desired dosage may be delivered morethan three times per day, three times per day, two times per day, onceper day, every other day, every third day, every week, every two weeks,every three weeks, every four weeks, every two months, every six months,or every twelve months. In some embodiments, the desired dosage may bedelivered using multiple administrations (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, ormore administrations).

Prophylactic Applications

In some embodiments, PNA agents in accordance with the invention may beutilized for prophylactic applications. In some embodiments,prophylactic applications involve systems and methods for preventing,inhibiting progression of, and/or delaying the onset of cancer or otherdisorder, and/or any other gene-associated condition in individualssusceptible to and/or displaying symptoms of cancer or other disorder.

Combination Therapy

It will be appreciated that PNA agents and therapeutically activeconjugates thereof in accordance with the present invention and/orpharmaceutical compositions thereof can be employed in combinationtherapies to aid in diagnosis and/or treatment. “In combination” is notintended to imply that the agents must be administered at the same timeand/or formulated for delivery together, although these methods ofdelivery are within the scope of the invention. Compositions can beadministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutics or medical procedures. In will be appreciatedthat therapeutically active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, each agent will be administeredat a dose and/or on a time schedule determined for that agent.

The particular combination of therapies (e.g., therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat pharmaceutical compositions of the PNA agents disclosed herein canbe employed in combination therapies (e.g., combination chemotherapeutictherapies), that is, the pharmaceutical compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutic and/or chemotherapeutic procedures.

PNA agents, or a pharmaceutically acceptable composition thereof, may beadministered in combination with chemotherapeutic agents to treatprimary or metastatic cancer. In some embodiments, an active ingredientis a chemotherapeutic agent, such as, but not limited to, Adriamycin,dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan,taxol, interferons, platinum derivatives, taxane (e.g., paclitaxel),vinca alkaloids (e.g., vinblastine), anthracyclines (e.g., doxorubicin),epipodophyllotoxins (e.g., etoposide), cisplatin, methotrexate,actinomycin D, actinomycin D, dolastatin 10, colchicine, emetine,trimetrexate, metoprine, cyclosporine, daunorubicin, teniposide,amphotericin, alkylating agents (e.g., chlorambucil), 5-fluorouracil,campthothecin, cisplatin, metronidazole, imatinib, Gleevec™, sunitiniband Sutent® and combinations thereof.

In some embodiments, PNA agents, conjugates thereof, or apharmaceutically acceptable composition thereof, are administered incombination with an antiproliferative or chemotherapeutic agent selectedfrom any one or more of Abarelix, aldesleukin, Aldesleukin, Alemtuzumab,Alitretinoin, Allopurinol, Altretamine, Amifostine, Anastrozole, Arsenictrioxide, Asparaginase, Azacitidine, BCG Live, Bevacuzimab, Avastin,Fluorouracil, Bexarotene, Bleomycin, Bortezomib, Busulfan, Calusterone,Capecitabine, Camptothecin, Carboplatin, Carmustine, Celecoxib,Cetuximab, Chlorambucil, Cisplatin, Cladribine, Clofarabine,Cyclophosphamide, Cytarabine, Dactinomycin, Darbepoetin alfa,Daunorubicin, Denileukin, Dexrazoxane, Docetaxel, Doxorubicin (neutral),Doxorubicin hydrochloride, Dromostanolone Propionate, Epirubicin,Epoetin alfa, Erlotinib, Estramustine, Etoposide Phosphate, Etoposide,Exemestane, Filgrastim, floxuridine fludarabine, Fulvestrant, Gefitinib,Gemcitabine, Gemtuzumab, Goserelin Acetate, Histrelin Acetate,Hydroxyurea, Ibritumomab, Idarubicin, Ifosfamide, Imatinib Mesylate,Interferon Alfa-2a, Interferon Alfa-2b, Irinotecan, Lenalidomide,Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Lomustine,Megestrol Acetate, Melphalan, Mercaptopurine, 6-MP, Mesna, Methotrexate,Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone,Nelarabine, Nofetumomab, Oprelvekin, Oxaliplatin, Paclitaxel,Palifermin, Pamidronate, Pegademase, Pegaspargase, Pegfilgrastim,Pemetrexed Disodium, Pentostatin, Pipobroman, Plicamycin, PorfimerSodium, Procarbazine, Quinacrine, Rasburicase, Rituximab, Sargramostim,Sorafenib, Streptozocin, Sunitinib Maleate, Talc, Tamoxifen,Temozolomide, Teniposide, VM-26, Testolactone, Thioguanine, 6-TG,Thiotepa, Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin,ATRA, Uracil Mustard, Valrubicin, Vinblastine, Vincristine, Vinorelbine,Zoledronate, or Zoledronic acid.

The particular combination of therapies to employ in a combinationregimen will generally take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved. It will also be appreciated that the therapies and/orchemotherapeutics employed may achieve a desired effect for the samedisorder (for example, an inventive antigen may be administeredconcurrently with another chemotherapeutic or neurological drug), orthey may achieve different effects. It will be appreciated that thetherapies employed may achieve a desired effect for the same purpose(for example, PNA agents useful for treating, preventing, and/ordelaying the onset of cancer or other disorder may be administeredconcurrently with another agent useful for treating, preventing, and/ordelaying the onset of cancer or disorders), or they may achievedifferent effects (e.g., control of any adverse effects). The inventionencompasses the delivery of pharmaceutical compositions in combinationwith agents that may improve their bioavailability, reduce and/or modifytheir metabolism, inhibit their excretion, and/or modify theirdistribution within the body.

In some embodiments, agents utilized in combination will be utilized atlevels that do not exceed the levels at which they are utilizedindividually. In some embodiments, the levels utilized in combinationwill be lower than those utilized individually.

In some embodiments, combination therapy may involve administrations ofa plurality of PNA agents directed to a single gene. In someembodiments, combination therapy can comprise a plurality of PNA agentsthat recognize distinct gene sequences.

Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods in accordance with the presentinvention. Kits typically comprise one or more PNA agents.

In some embodiments, kits for use in accordance with the presentinvention may include one or more reference samples; instructions (e.g.,for processing samples, for performing tests, for interpreting results,for administering PNA agents, for storage of PNA agents, etc.); buffers;and/or other reagents necessary for performing tests. In someembodiments kits can comprise panels of PNA agents. Other components ofkits may include cells, cell culture media, tissue, and/or tissueculture media.

In some embodiments, kits include a number of unit dosages of apharmaceutical composition comprising PNA agents. A memory aid may beprovided, for example in the form of numbers, letters, and/or othermarkings and/or with a calendar insert, designating the days/times inthe treatment schedule in which dosages can be administered. Placebodosages, and/or calcium dietary supplements, either in a form similar toor distinct from the dosages of the pharmaceutical compositions, may beincluded to provide a kit in which a dosage is taken every day.

Kits may comprise one or more vessels or containers so that certain ofthe individual components or reagents may be separately housed. Kits maycomprise a means for enclosing the individual containers in relativelyclose confinement for commercial sale, e.g., a plastic box, in whichinstructions, packaging materials such as styrofoam, etc., may beenclosed.

In some embodiments, kits are used in the treatment, diagnosis, and/orprophylaxis of a subject suffering from and/or susceptible to cancer orother disorder. In some embodiments, such kits comprise (i) at least onePNA agent; (ii) a syringe, needle, applicator, etc. for administrationof the at least one PNA agent to a subject; and (iii) instructions foruse.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES

General Details

Example 1: Inhibition of BRAF Expression Using Peptide Nucleic Acids(PNA)

Cell Culture

The present example demonstrates the effect of BRAF mutant specific PNAderivatives cell viability. PNA compounds targeting the BRAF V600E genemutation were tested against melanoma cell lines that are either BRAFwild type (C918) or BRAF V600E mutant (OCM1A and SK-MEL 7). C918 andOCM1A are uveal melanoma cell lines while SK-MEL 7 is a cutaneousmelanoma cell line. C918 was kindly provided by Robert Folberg(University of Illinois, Chicago, Ill.). OCM1A was from Dr. WilliamHarbour (Washington University, St. Louis, Mo.). SK-MEL 7 was from AlanHoughton (Memorial Sloan-Kettering Cancer Center, New York, N.Y.). Cellswere cultured in RPMI medium supplemented with 10% FBS, 100 units/mLpenicillin, and 100 mg/mL streptomycin and maintained at 37° C. in 5%CO₂.

Cell Viability Assays

PNA compounds targeting the BRAF V600E gene mutation were tested againstmelanoma cell lines that are either BRAF wild-type (C918) or BRAF V600Emutant (OCM1A and SK-MEL 7). C918 and OCM1A are uveal melanoma celllines while SK-MEL 7 is a cutaneous melanoma cell line. Two PNAderivatives in particular, (A) I-292-3 L2LP NHAc and (B) I-292-9 L2NHAc, showed significant suppression of cell viability and specificityfor the cell lines that have the target gene mutation, OCM1A andSK-MELT. Cells were treated with increasing doses of several PNAderivatives for 72 hours after which the cell viability was measured andcalculated relative to the untreated cells. Cells were plated in 96-wellplates and treated in triplicates with the indicated concentrations ofPNA derivative. Viability was assessed after 72 hours of treatment usingthe Cell Counting Kit 8 (CCK8) from Dojindo Molecular Technologiesaccording to the manufacturer's instructions. Survival is expressed as apercentage of untreated cells—displaying stagnant uptake <10% ID/g atall timepoints (24-120 hours p.i.), possibly due to enhanced permeationand retention of the tumor's leaky vasculature. As depicted in FIGS.2A-2B, cells treated with the PNA derivatives I-292-3 L2LP NHAc (as seenin FIG. 2A) and I-292-9 L2 NHAc (as seen in FIG. 2B) showed significantsuppression of cell viability and specificity for the cell lines (OCM1Aand SK MEL 7) that have the target gene mutation. The PNA derivativeI-292-3 L2LP NHAc inhibited SK MEL 7 cell growth by 100%. The PNAderivative I-292-3 L2LP NHAc inhibited OCM1A cell growth by 96.7%.Incubation of SK MEL 7 and OCM1A cells with I-292-3 L2LP NHAc caused a62.4% and 40.3% (respectively) decrease in BRAF expression, asdetermined by Western blot. Specificity is seen at lower doses in whichcell viability of mutant cells was reduced to 20% or less with 750 nMtreatment of the PNA derivative, while the viability of C918 cellsremained around 100%. This demonstrates that the PNA derivatives areeffective at suppressing cell viability and are specific for genes withthe target BRAF mutation.

Immunoblotting

Cells were treated for 24, 48 and 72-hour treatments with 750 nM PNA andlysed in radioimmunoprecipitation assay (RIPA) buffer supplemented withprotease inhibitor cocktail tablets (Roche Diagnostics) and 1 mmol/LNa3VO4. Equal amounts of protein were loaded on 4% to 12% PAGE gels(Invitrogen). Polyvinylidene difluoride (PVDF) membranes were blockedwith 5% nonfat dried milk and probed with p-MEK, MEK, p-ERK 1/2(T202/Y204), ERK 1/2, cleaved PARP, α-tubulin (Cell SignalingTechnology), BRAF (Santa Cruz Biotechnology), LC3 (Novus Biologicals)and BRAF V600E (Spring Bioscience).

FIGS. 3A-3C depict BRAF mutant and BRAF wild type protein expression inmelanoma cells (C918 and OCM1A) treated with 750 nM of the PNA I-292-3L2LP NHAc for 24, 48 or 72 hours. FIG. 3A depicts the measurement ofmutant and total BRAF protein expression as measured by Western blottingin C918 and OCM1A cells. Tubulin expression was also measured as aloading control. Wild type BRAF is found in both wild type and mutantcell lines while the BRAF V600E mutant was only found in mutant cells.Mutant BRAF protein expression is reduced over time in cells treatedwith the PNA derivative. No mutant BRAF protein was observed in C918cells as it is a wild-type cell line. FIG. 3B depicts decreasedexpression of BRAF V600E mutant protein in OCM1A (BRAF mutant) celllines over 72 hour of treatment with the PNA derivative I-292-3 L2LPNHAc. No mutant BRAF expression was seen in C918 cells. FIG. 3C showsthat there was no significant reduction of total BRAF wild type proteinover 72 hours in either OCM1A (mutant) and C918 (wild type) cellstreated with I-292-3 L2LP NHAc. These results correlate with thespecificity and viability suppression seen in the cell viabilitystudies. FIGS. 3A-3C demonstrate that PNA derivatives are effective atsuppressing cell viability and are specific for genes with the targetBRAF mutation. ND=untreated.

FIGS. 4A-4C depict BRAF mutant and BRAF wild type protein expression inmelanoma cells (C918 and OCM1A) treated with 750 nM of the PNA I-292-9L2 NHAc for 24, 48 or 72 hours. FIG. 4A depicts the measurement ofmutant and total BRAF protein expression as measured by Western blottingin C918 and OCM1A cells. Tubulin expression was also measured as aloading control. Wild type BRAF is found in both wild type and mutantcell lines while the BRAF V600E mutant is only found in mutant cells.Mutant BRAF protein expression is reduced over time in cells treatedwith the PNA derivative. No mutant BRAF protein was observed in C918cells as it is a wild-type cell line. FIG. 4B depicts decreasedexpression of BRAF V600E mutant protein in OCM1A (BRAF mutant) celllines over 72 hours of treatment with the PNA derivative I-292-9 L2NHAc. No mutant BRAF expression was seen in C918 cells. FIG. 4C showsthat there was no significant reduction in total BRAF wild type proteinover 72 hours in C918 (wild type) cells treated with I-292-9 L2 NHAc.There was a slight decrease of total BRAF protein expression in 0C1MA(mutant) cells, but not in C918 (wild type) cells. These results alsocorrelate with the specificity and viability suppression seen in thecell viability studies. FIGS. 4A-4C demonstrate that PNA derivatives areeffective at suppressing cell viability and are specific for genes withthe target BRAF mutation. ND=untreated.

Quantitative real-time PCR

C918 and OCM1A cells were treated with 750 nM PNA (I-292-3 L2LP) for 48hours. Mutant BRAF mRNA expression in cells was quantified using RT-PCT.Specifically, cells were treated and then lysed using TRIzol Reagent®(Invitrogen). Reverse transcription of 1 μg of RNA was done using theSuperScript III First-Strand Synthesis System (Invitrogen). Quantitativereal-time PCR (qRT-PCR) assays were performed using the 7300 Real TimePCR System (Applied Biosystems). TaqMan gene expression assays, whichinclude gene-specific probe primer sets (Applied Biosystems), were usedto detect the indicated genes and glyceraldehyde-3-phosphatedehydrogenase (GAPDH)/hypoxanthine phosphoribosyltransferase (HPRT)mRNA. The relative expression of each gene was calculated by the ΔΔC_(T)method. See FIGS. 5A-5B. Cells wild-type for BRAF (C918) as well asmutant cell lines (OCM1A and SK-Mel 7) were treated for 48 hours with750 nM I-292-3 L2LP NHAc. According to FIG. 5A, BRAF^(V600E) mRNAexpression was quantified using RT-PCR. Relative Quantity (RQ) valueswere normalized with GAPDH loading control and set relative to themutant BRAF mRNA expression in untreated OCM1A cells (ND). BRAF^(V600E)mRNA expression significantly decreased in OCM1A cells when treated withI-292-3 L2LP NHAc. According to FIG. 5B, Both BRAF^(V600)E and totalBRAF mRNA expression in SK-Mel 7 cells were quantified using RT-PCR. RQvalues were normalized with GAPDH and set relative to untreated cells.In SK-Mel 7 cells, BRAF^(V600E) mRNA expression also decreasedsignificantly after treatment with I-292-3 L2LP NHAc. There was noobserved decrease in total BRAF mRNA expression as a result oftreatment. The RT-PCT results for mutant BRAF mRNA expression wereconsistent with what could be seen in Westerns blots showing decreasedprotein expression in FIGS. 3A-3C and 4A-4C.

Xenograft Mouse Trials

Athymic mice (3 per group) were inoculated with OCM1A. After 15 days,the size of the developing tumor was sufficient for further measurementand monitoring. The treatment group was given 50 mg/kg dosage of I-292-3L2LP-NHAc on days 15, 17, 20 and 22. As seen in FIG. 6A, on day 28, theaverage tumor size was 35% that of the untreated control group.Moreover, one week following the last dose, the average tumor size ofthe treated group began to shrink by 25% over 4 days.

Toxicity of the tested PNA derivative was evaluated by measuring weightloss of test animals. At the dosages used, animal weight and activitydid not change throughout the 28 day study, as seen in FIG. 6B. Theseresults correlate well with the lack of toxicity observed in previous invitro studies.

The Memorial Sloan-Kettering Cancer Center Institutional Animal Care andUse Committee and Research Animal Resource Center specifically approvedthis study. The study also complied with the principles of LaboratoryAnimal Care (NIH publication no. 85-23, released 1985). All efforts weremade to minimize animal suffering.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description.Likewise, those of ordinary skill in the art will readily appreciatethat the foregoing represents merely certain preferred embodiments ofthe invention. Various changes and modifications to the procedures andcompositions described above can be made without departing from thespirit or scope of the present invention, as set forth in the followingclaims.

In the claims articles such as “a”, “an” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Thus, for example, reference to “an antibody” includes aplurality of such antibodies, and reference to “the cell” includesreference to one or more cells known to those skilled in the art, and soforth. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are presenting, employed in, or otherwise relevant to agiven product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitation, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim. For example, any claim that is dependent on another claim can bemodified to include one or more limitations found in any other claimthat is dependent on the same base claim. Furthermore, where the claimsrecite a composition, it is to be understood that methods of using thecomposition for anyone of the purposes disclosed herein are included,and methods of making the composition according to any of the methods ofmaking disclosed herein or other methods known in the art are included,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldbe understood that, in general, where the invention, or aspects of theinvention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is noted that the term“comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understand of one of ordinary skill in the art, values thatare expressed as ranges can assume any specific value or sub-rangewithin the state ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention can beexcluded from any one or more claims, for any reason, whether or notrelated to the existence of prior art.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

What is claimed is:
 1. A method of reducing expression of a target genein a cell comprising: contacting a cell in which the target is expressedwith at least one PNA agent, wherein the PNA agent comprises a firstcationic and/or hydrophobic moiety at a first end of the PNA moiety; anda second cationic and/or hydrophobic moiety at a second end of the PNAmoiety; determining a level or activity of the target in the cell whenthe PNA agent is present as compared with a target reference level oractivity observed under otherwise comparable conditions when it isabsent; wherein the level or activity of the target is reduced when thePNA agent is present as compared with the target reference level oractivity, thereby reducing expression of the target gene.
 2. The methodof claim 1, wherein the first peptide comprises lysine residues, andoptionally at least one of the lysine residues comprises a palmitoylside chain moiety.
 3. The method of claim 1, wherein the second peptidecomprises lysine residues, and optionally at least one of the lysineresidues comprises a palmitoyl side chain moiety.
 4. The method of claim1, wherein the first peptide and/or the second peptide consists oflysine residues.
 5. The method of claim 1, wherein the PNA agent has asequence that contains less than 60% purines.
 6. The method of claim 1,wherein the PNA moiety has a sequence that targets a gene.
 7. The methodof claim 1, wherein the PNA moiety has a sequence that targets a 13-20nucleotide sequence of a gene with 75% or greater complementarity. 8.The method of claim 1, wherein the PNA moiety has a sequence thattargets a 13-20 nucleotide sequence of a gene with completecomplementarity.
 9. The method of claim 6, wherein the gene is anoncogene.
 10. The method of claim 9, wherein the PNA agent has asequence that targets a mutation in the oncogene.
 11. The method ofclaim 6, wherein the PNA agent targets a site selected from the groupconsisting of: a) a region of a BRAF oncogene; b) a region of a BRAFoncogene corresponding to a V600E mutation in a BRAF protein; or c) aregion of a Gnaq gene corresponding to a Q209L mutation in a Gnaqprotein.
 12. The method of claim 9, wherein the PNA agent targets aregion comprising a translocation junction of an oncogene.
 13. Themethod of claim 6, wherein the PNA agent targets a site comprising aregion of: a) a MYB-NFIB translocation comprising a junction of MYB andNFIB genes or fragments thereof; b) a FUS-CHOP translocation comprisinga junction of FUS and CHOP genes or fragments thereof; c) a EWS-FLITtranslocation comprising a junction of EWS and Fill genes or fragmentsthereof; d) a BCR-ABL translocation comprising a junction of BCR and ABLgenes or fragments thereof; or e) a SYT-SSX translocation comprising ajunction of SYT and SSX genes or fragments thereof.
 14. The method ofclaim 9, wherein the oncogene is selected from the group consisting ofABL1, ABL2, ALK, AKT1, AKT2, ATFL, BCL11A, BCL2, BLC3, BCL6, BCR, BRAF,CARD11, CBLB, CBLC, CCND1, CCND2, CCND3, CDX2, CTNNB1, DDB2, DDIT3,DDX6, DEK, EGFR, ELK4, ERBB2, ETV4, ETV6, EVIL EWSR1, FEV, FGFR1,FGFR1OP, FGFR2, FUS, GOLGA5, HMGA1, HMGA2, HRAS, IRF4, IDH1, IDH2, JUN,KIT, KRAS, LCK, LMO2, MAF, MAFB, MAML2, MDM2, MET, MITF, MLL, MPL, MYB,MYC, MYCL1, MYCN, NCOA4, NFKB2, NRAS, NTRK1, NUP214, PAX8, PDGFB,PIK3CA, PIM1, PLAG1, PPARG, PTPN11, RAFT, REL, RET, ROS1, SMO, SS18,TCL1A, TET2, TFG, TLX1, TPR, and USP6.
 15. The method of claim 1,wherein the cell is in a subject having or risk of having a disease,disorder, or condition and administration of the PNA agent to thesubject treats or reduces the risk of the disease, disorder, orcondition in the subject.
 16. The method of claim 15, wherein thedisease, disorder or condition is cancer or a tumor.
 17. The method ofclaim 16, wherein the cancer or tumor is selected from prostate cancer,melanoma, ocular melanoma and/or sarcoma.
 18. The method of claim 15,wherein the first peptide comprises lysine residues, and optionally atleast one of the lysine residues comprises a palmitoyl side chainmoiety.
 19. The method of claim 15, wherein the second peptide compriseslysine residues, and optionally at least one of the lysine residuescomprises a palmitoyl side chain moiety.
 20. The method of claim 15,comprising further administration of a treatment modality selected fromradiation, chemotherapy or surgery.